Real time multipoint assay for optimizing performance

ABSTRACT

Described herein are methods of optimizing the performance of a mammalian semen sample with respect to outcomes such as fertility, using a multipoint assay to determine an optimum time point for preparing the semen sample for use in insemination. The multipoint assay is based upon a kinetic model of biomarker expression during sperm capacitation relative to the outcome of interest.

RELATED APPLICATIONS

This application is a continuation-in-part of PCT international application Serial No. PCT/US2010/050881, filed Sep. 30, 2010, which is a continuation in part of U.S. Ser. No. 12/571,361, filed Sep. 30, 2009, which is a continuation in part of PCT/US09/39022, filed Mar. 31, 2009, which claims the benefit of U.S. provisional application No. 61/040,717, filed Mar. 31, 2008, and U.S. provisional application No. 61/040,798 filed Mar. 31, 2008. The contents of each of these applications are incorporated herein by reference in their entirety.

BACKGROUND

Treatment of mammalian semen to achieve a higher proportion of fertility and/or a higher proportion of one gender over another in artificial insemination can be advantageous. For example, a dairy herd would obtain economic and genetic herd quality benefit from an increase in numbers of cows pregnant at any given time and/or birthing a higher percentage of heifers relative to bulls. In such a situation, replacement animals for the herds are produced more efficiently. In addition, especially with low-beef value animals such as Holsteins, the expense of bull calves, and the potential cruelty these animals face when used in veal production is reduced.

The availability of replacement female animals born at the dairy farm eliminates the need to import replacements and the attendant risk of disease introduction into a herd. Additional advantages are found for businesses housing elite sires that produce dairy bull semen. Since these bulls are evaluated, i.e. “sire-proofed,” for genetic quality through their daughters, an elite bull can be brought into semen production more quickly if he produces daughters more quickly and often. This speeds improvement of the sire genotype, with the attendant competitive advantage. This further produces a savings in feed, vetinarian care, and other costs associated with bull farming. It also accelerates the improvement of the genetic base of dairy herds using semen from these processors, with the attendant economic savings to dairy farmer and semen processor alike.

In addition, achieving good fertility by increasing the quality of sperm used in artificial insemination is considered to be the single greatest determinant of the success or failure of dairy farms. Since “open” or non-pregnant cows do not lactate and are therefore not productive, they decrease profit. Consequently, any increase in fertility is considered worthwhile. Fertility is important for all types of animals raised for dairy or for meat such as goats, sheep, cattle, buffalo, camels, swine, etc.

In another example, increased sperm quality can lead to improvement and/or expansion of a particular population of animals. For instance, sperm collected from elite race horses or other champion animals, such as cattle or other livestock and particular breeds of dogs and cats, is commonly used for artificial insemination to increase the probability of maintaining particular features in the gene pool. Sperm quality is particularly important in the breeding programs directed to exotic and endangered animals where the number of captive individuals is limited. Here, the ability to increase overall birth rates, thereby increasing the potential for rapid expansion of the population, is critical for success.

In another example, the personal suffering and costs associated with human infertility can in many cases be reduced through increasing sperm quality. Couples whose infertility is caused by low sperm count or poor sperm motility can benefit by increasing the number of intact and viable sperm that result after the washing and preparations steps needed prior to intrauterine artificial insemination (NI) or intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF).

With respect to gender bias, the suffering and costs of human sex-linked diseases can be reduced through birth of females in affected human families. Female births are the only way to eliminate over 300 X-linked diseases, many of which shorten and impair quality of life and create staggering medical costs. Currently, the costs and suffering associated with these diseases can be decreased through pre-implantation genetic diagnosis. In this process, eggs are harvested by laparoscopy following injections of hormones and fertility drugs. Eggs are fertilized in vitro and, after embryos have reached sufficient size, a single cell is microdissected from each embryo for genetic analysis. A suitable unaffected female embryo is chosen for implantation. Alternatively, sperm is collected and treated with mutagenic dye in preparation for fluorescent activated cell sorting (FACS). X-bearing sperm are obtained, however, they are so damaged that the sperm nucleus must be injected into an isolated egg in vitro using intracytoplasmic egg injection. Embryos are then cultured and implanted in recipients. Both of these techniques are expensive and raise unresolved questions about the effect of either hormonal treatments of the recipient or of exposure to DNA-binding dyes and laser light, with respect to their cytotoxicity and mutagenic potential (Downey et al. (1991) J. Histochem. and Cytochem. 39: 485-489; Durand and Olive (1982) J. Histochem. and Cytochem. 30:111-116).

The scientific literature describes several methods for achieving gender bias through treatment of mammalian semen. They differ in process; some involve physical separation of sperm while others do not. They also differ at point of application; to sperm, to female mammals, to clutches of eggs in egg-laying animals. What they share in common is that they cannot be applied effectively on-site. In addition, with the exception of the instant invention, there is no technology readily used for insemination of cows. Fertility issues with other technologies restrict their use to virgin heifers, which are less stressed and therefore have higher fertility than cows that have experienced the stress of lactation.

For example, several methods are reported for generating sex bias by physical separation of sperm, all of which involve complex laboratory manipulations and equipment. Fluorescence activated cell sorting (FACS) resolves sperm into X (female) and Y (male) bearing pools, after cell labeling with mutagenic DNA-binding dyes to reveal chromosome content (Abeydeera et al. (1998) Theriogenology 50: 981-988; Cran and Johnson (1996) Human Reproduction Update 2: 355-363). Methods of artificially biasing the sex of mammalian offspring through physical separation have also included methods based upon density sedimentation of spermatozoa (e.g. Brandriff et al. (1986) Feral. Steril. 46:678-685) and by separating the population of spermatozoa into fractions that differ by the sex-linked electrical charge resident thereon (U.S. Pat. No. 4,083,957). Methods have also been described that rely on mechanical sorting of sperm by sex-type. U.S. Pat. No. 5,514,537, for example, uses a column packed with two sizes of beads. The large beads are of a diameter so that the smaller beads will fall between the interstices created between the larger beads. Then the interstices between the smaller beads allow Y-bearing sperm to enter them while the X-bearing sperm are excluded, thereby effecting separation of the two subpopulations. Separation based on immunological methods and cell surface markers have also been proposed (Blecher et al. (1999) Theriogenology 52: 1309-1321). In another example, U.S. Pat. No. 3,687,806 discloses an immunological method for controlling the sex of mammalian offspring using antibodies that react with either X-bearing sperm or Y-bearing sperm which uses an agglutination step to separate bound antibodies from unaffected antibodies. U.S. Pat. No. 4,191,749 discloses a method for increasing the percentage of mammalian offspring of either sex by using a male-specific antibody coupled to a solid-phase immunoabsorbant material to selectively bind male-determining sperm while female-determining sperm remain unbound in a supernatant. U.S. Pat. No. 5,021,244 discloses a method for sorting living cells based upon DNA content, particularly sperm populations to produce subpopulations enriched in X-bearing sperm or Y-bearing sperm by means of sex-associated membrane proteins and antibodies specific for such proteins.

Some methods have combined various aspects of the immunological and mechanical separations such as U.S. Pat. Nos. 6,153,373 and 6,489,092 which use antibodies coupled to a magnetic particle for separation of sperm.

Separation based on a miniscule size difference between X- and Y-bearing sperm has also been attempted (Van Munster et al. (1999) Theriogenology 52: 1281-1293; Van Munster (1999) Cytometry 35: 125-128; Van Munster 2002 Cytometry 47: 192-199).

In addition, sex bias without physical separation of sperm into X and Y bearing classes has been described. For example, stress (Catalano et al. (2006) Human Reproduction 21: 3127-3131), good or poor physical condition (Trivers and Willard (1973) Science 179:90-92), feed composition (Alexenko et al. (2007) Biol. Reprod. 77:599-604), temperature (Crews (1996) Zoological Science 13: 1-13) and other factors (Wedekind (2002) Animal Conservation 5:13-20) have been shown to affect offspring sex ratio.

Lechniak (2003, Reprod. Dom. Anim. 38:224-227); has also shown that time-based production of a sex bias sexing of semen can occur when semen is held for various times before use in insemination for in vitro fertilization. However, the exact time course of activation of sperm from its dormant state at the time of collection, through its various metabolic states of fertility, until the sperm finally become infertile and atrophied, varies between different species of mammals, and also between different individuals of the same species, and even between ejaculates obtained from the same individual animal.

This large degree of variability in time course from semen samples collected from the same individual led those skilled in the art to conclude that a fertile semen sample having a gender bias could not be reliably obtained simply by processing a sample for insemination after a standard period of time after collection of the semen sample. Therefore, there is a need in the art to develop a time based assay on which one can reliably depend to provide a semen sample containing sperm which have a desirable trait, such as a fertile, gender biased, semen sample. Ideally, the assay could be performed on site.

Sperm become able to fertilize—capacitate—at wide-ranging times spanning hours that are unique to each ejaculate. Semen testing is not done at the same time as insemination or as freezing doses of sperm, meaning the status of the sperm at the time of insemination is not known. This is one reason semen tests do not correlate with fertility. A single-point assay of semen may indicate poor quality, when it may have simply been tested too early. Conversely, the semen may test well but be past its prime at insemination or freezing. This can occur because (1) single point assays do not identify the optimal state of sperm and (2) therefore, sperm cannot be stabilized in the optimal state.

One in six couples is affected by reproductive issues, including infertility. Many interventions exist for female-factor infertility, but male-factor infertility has few good options available. Sperm assays exist, but people are pessimistic about their utility. This is understandable as explained above, because the assays currently in use take a photograph of the sperm, i.e., a snapshot in time.

These assays are not applied to ejaculates in real time, that is immediately post-collection and at repeated time points,—to reveal the dynamic and changing nature of sperm. These changes include acquisition, at a time unique to each ejaculate—of abilities such as fertilizing ability, or of reaching the state of maximal fertility for that ejaculate, or of ability to successfully resist damage from processes such as freezing and vitrification, or of ability to produce gender bias, the gender bias being useful for example, in dairy cattle calvings. Only very fast assays can do this—run as multipoint assays starting immediately after ejaculation and repeated during the hours that mammalian sperm undergo maturation prior to insemination. Fast assays applied this way (according to Applicant's novel methods and products described herein) enable optimization of sperm properties by customizing sperm handling to the unique timing of every ejaculate's sperm maturation. To accomplish such a goal requires a fast assay that produces—not a snapshot—but a movie.

Evaluating a semen sample according to the real time methods described herein enables optimizing the timing for processing a semen sample for insemination according to the desired performance of the sample, for example increased fertility. In these embodiments of the invention, semen samples can be obtained which have increased numbers of inseminations per number of sperm cells, increased number of straws per ejaculate, greater viability post-thaw of frozen semen doses, and/or higher fertility at same cell number.

A real-time assay that reports fertilizing ability would allow medical personnel to select the optimal status of sperm to best enable fertility. Described herein is an assay that detects the dynamic changes in sperm in real time, so that the optimal cell status can be selected prior to insemination or freezing. Applicant's novel disclosure herein also teaches a means of stabilizing the sperm to retain this optimal condition. In this way, for the first time to Applicant's knowledge, the full potential of the sperm in every ejaculate can be realized.

SUMMARY

It is recognized in the art that sperm are infertile when ejaculated, and that they only gain the ability to fertilize over time, as they mature. The time for this gain is unique to each freshly obtained ejaculate. Accordingly, clinical assays that evaluate sperm at a single point in time, thereby fail to detect the time of fertility acquisition—or loss thereof as sperm age—unique to each ejaculate's maturation and sensecence. These assays therefore do not show the maximum potential for each collection or the optimal point for improvement of male-side infertility interventions. As a result, perfectly functional ejaculates may be misidentified as subfertile, or may be inseminated when they are not optimally fertile. Infertility may be exacerbated in the clinic in the absence of a way to optimize sperm fertility.

The assays of the instant invention overcome obstacles to male side infertility interventions by accommodating the variability in the timing by sperm from different semen samples in gaining and reaching maximum of the ability to fertilize. Improved fertility as well as other desirable traits have been produced with Applicant's methods and products disclosed herein, including the first real time, multipoint assay enabling optimization of sperm fertilizing ability (maturational state) before insemination. A variant of Applicant's approach for dairy cattle produces increased female calvings or increased male calvings, as also described herein.

The present invention provides methods of process control for processing semen into doses at AI stations that house elite-dairy bull sires. These methods enable more consistent results for selection of field traits previously desired but unattainable without this process control. The methods include monitoring of the in-process product—collected semen—in real time for processing into finished product (dairy farmers receive semen as individual cow-doses) at the AI station. The present invention provides a solution to the impediment to desired results because of variability in the metabolic rates of sperm activation, which has hindered the development of a time based assay for obtaining a semen sample having a desired trait, e.g., fertile gender biased sperm. Specifically, the methods described herein involve monitoring, in real time, changes in the metabolic status of sperm in a semen sample. A real time multipoint assay of the metabolic status of sperm in an individual semen sample allows a better, more consistent determination of when, after collection of an individual sperm sample, the sperm in the semen sample have the desired traits, e.g., fertility, gender bias. The real time multipoint monitoring of the metabolic status of sperm in a semen sample as it progresses through its individual metabolic rate of sperm activation, for example, can be used to determine the real time occurrence of expression of a desired trait in an individual semen sample, such as optimum fertility and/or a gender bias. Advantageously, the methods disclosed herein can be used on site, and are gentle enough that the product can be used to inseminate cows without a loss of fertility that would render the process economically nonviable.

In one embodiment there is a method for obtaining a semen sample with sperm having a desired trait comprising monitoring the metabolic status of said sperm in the sperm sample during incubation, the method comprising the steps of: i) selecting a marker that can be indicative of a metabolic status of sperm, wherein expression of the marker changes during said incubation; ii) determining the level of expression of the marker by sperm of the semen sample at a plurality of time points during said incubation, wherein optionally an aliquot of said sample is assayed at each time point; iii) determining the time point at which the sperm in an aliquot of the semen sample express the marker at about a predetermined level; and iv) processing said semen sample at a time later than the time point in previous step (iii), wherein said later time is determined by adding a time shift previously established for said marker to the time at the time point determined in previous step (iii), thereby obtaining sperm with the desired trait.

In another embodiment there is a method for determining a time shift for processing a semen sample by monitoring a change in the metabolic status of sperm in the semen sample during incubation, the method comprising the steps of: i) selecting a marker that can be indicative of a metabolic status of sperm, wherein expression of the marker changes during said incubation, ii) determining the level of expression of the marker by sperm of the semen sample at a plurality of time points during said incubation, wherein optionally an aliquot of said sample is assayed at each time point, iii) determining a time point when the sperm in the semen sample display a maximum level of a desired trait, iv) selecting a time point (jump point) which occurs before the sperm display the maximum level of the desired trait, v) determining the level of expression of the marker by sperm in said sample at the selected time point (jump point); vi) determining the elapsed time between the time point of step (iii) and step (iv), thereby providing a time shift for a change in the metabolic status between the jump point and the maximum level of the desired trait, as reflected by the marker, wherein other semen samples can be processed for artificial insemination based on monitoring the marker to the jump point and applying the time shift to determine the desired period for incubation.

In one embodiment there is a method for obtaining a semen sample with sperm having a desired trait comprising monitoring the metabolic status of said sperm in the sperm sample during incubation, the method comprising the steps of: i) selecting a marker that can be indicative of a metabolic status of sperm, wherein expression of the marker changes during said incubation; ii) determining the level of expression of the marker by sperm of the semen sample at a plurality of time points during said incubation, wherein optionally an aliquot of said sample is assayed at each time point; iii) determining the time point at which the sperm in an aliquot of the semen sample express the marker at about a level represented by the jump point of; and iv) processing said sample at a later time, wherein said later time is determined by adding the time shift established for the marker, to the time at the time point determined in the immediately previous step (iii); thereby obtaining sperm with the desired trait.

In yet another embodiment there is a method for determining the jump point for a particular marker for the metabolic status of sperm in a semen sample during incubation, comprising the steps of: i) determining the change in percentage of the sperm displaying a marker/indicator of the metabolic status over time during the incubation or the condition under which the sperm is being held; ii) determining when the sperm in the semen sample display significant fertility and/or a significant a significant gender bias and/or a desired trait, where in one embodiment, the sperm in the sample will be capable of producing the desired trait(s) upon artificial insemination in the field; iii) selecting a time point (jump point) which occurs before the sperm display a desired trait, and iv) determining the percentage of sperm in said sample which display said marker/indicator at the selected time point (jump point); thereby monitoring a change in the metabolic status of said sperm in said sample during said incubation, and further determining the jump point for each marker of metabolic status.

In another embodiment, a method for obtaining a semen sample with sperm having a desired trait comprising monitoring the metabolic status of said sperm in the sperm sample during incubation, the method comprising the steps of: i) selecting a marker that can be indicative of a metabolic status of sperm, wherein expression of the marker changes during said incubation; ii) determining the level of expression of the marker by sperm of the semen sample at a plurality of time points during said incubation, wherein optionally an aliquot of said sample is assayed at each time point; iii) determining the time point at which the sperm in an aliquot of the semen sample express the marker at about a maximum level by noticing a drop in expression of said marker; and iv) processing said sample at that time; thereby obtaining sperm with the desired trait.

Thus, one embodiment described herein is a method for obtaining a semen sample collected, from a male animal, which contains an increased proportion of sperm having a desired trait, by monitoring the metabolic status of said sperm in a sperm sample, comprising the steps of: providing a semen sample, which in one aspect has been newly collected from a male, or in another aspect, is a sample which has been frozen or otherwise maintained after collection; assaying sperm of said sample over time for expression of a marker indicative of a metabolic stage of said sperm, where in one nonlimiting aspect the assay is performed on an aliquot of the semen sample; processing said sample at a predetermined later time point, wherein, at said later time point, the semen sperm of said sample have the desired trait; thereby obtaining a sample of sperm with the desired trait.

In one aspect of this embodiment, the desired trait includes, but is preferably not limited to, female gender bias of sperm of the semen sample, male gender bias of sperm of the semen sample, fertility of the semen in the semen sample, and/or a combination of fertility and gender bias, and or another trait. Performance also includes the ability of a semen sample to resist or withstand procedures for preparing the semen sample for insemination, including the procedures of freezing and vitrification. Performance also includes an optimal viability of sperm of a semen sample after thawing.

In another aspect of this embodiment, the marker includes, but is preferably not limited to, acrosome length or morphology, expression of a cell surface molecule of sperm of the semen sample, electrostatic charge of sperm of the semen sample, and permeability of a dye by sperm or fragments thereof of sperm of the semen sample.

In another aspect of this embodiment, the semen sample is assayed for said marker at intervals ranging from any of 1 to 120 minutes, including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110, 115 and 120 minutes. The interval used can depend on many factors, including, but preferably not limited to the rate of change of expression of the marker. In a further aspect, the assay may encompass the use of an aliquot of the semen sample.

In another aspect of this embodiment, the sperm sample is incubated at a constant temperature during said monitoring of the metabolic status.

In another aspect of this embodiment, the marker and/or biomarker is selected from, though preferably not limited to, a ligand, a lectin, an enzyme and a receptor, which is expressed on the surface of the sperm, or internally or both. In one embodiment, a ligand includes, but preferably is not limited to, a protein, a glycoprotein, a carbohydrate and a glycolipid. In another embodiment the marker is selected from, though preferably not limited to, acrosome length, acrosome morphology, acrosome ruffling, expression of a cell surface molecule, electrostatic charge of said sperm, and permeability of sperm membrane, a lipid, cholesterol, phosphatidylserine, a sugar and a protein, an intracellular ion, and bicarbonate.

In one embodiment binding of said first ligand evokes the appearance of a secondary marker, which is detected by contacting said secondary marker with a supplemental ligand.

In another nonlimiting aspect of this embodiment, the permeability of a dye by said sperm or fragments thereof is assayed by the intensity of punctate staining by said dye in an aliquot of said sperm sample.

In another aspect of this embodiment, the permeability of said sperm or fragments to a dye is monitored, and the time point selected to process the semen sample is determined with respect to the earlier time point when the sample has the determined intensity of punctate staining by a dye.

In another aspect of this embodiment, the expression of a cell surface marker is monitored, and the time point selected to process the semen sample is determined with respect to the earlier time point when the sample maximally expresses the marker.

In another aspect of this embodiment, the expression of a cell surface marker is monitored, and the time point selected to process the semen sample is determined with respect to the earlier time point when expression of said marker in said sample has decreased relative to its peak expression by a percentage preferably, but not limited to ranging from up to 99% to 5%, though any detectable amount of change in expression of any marker is encompassed by the assays described herein.

In another embodiment, the expression of a cell surface marker is monitored, and the time point selected to process the semen sample is measured with respect to the earlier time point when expression of said marker in said sample has increased from its minimal expression by a percentage increase ranging from 5% to 1000% when compared to a prior time point.

In another embodiment, the desired trait is an excess of active female sperm relative to active male sperm in said sample.

In another embodiment, the desired trait is an excess of male sperm relative to female sperm in said sample.

In another embodiment, the desired trait is sperm with optimal fertility.

In another embodiment, the desired trait is sperm which display resistance to freezing, vitrification, dehydration and other methods of sperm stabilization such as encapsulation of the semen sample or portion thereof.

In one embodiment, the assay is based on the percentage of sperm in the semen sample having a specified marker. In one aspect, the specified marker is a biochemical marker, which is optionally present on the cell surface. Regardless, the marker reflects or is indicative of the metabolic status of the sperm. The marker is not limited to a sperm specific marker.

In one aspect of this embodiment the assay encompasses determining the percentage of sperm in the semen sample having the marker that reflects the metabolic status of the sperm encompasses the steps of a) removing an aliquot from the semen sample; b) contacting the aliquot with a first ligand to said marker; c) detecting binding of said ligand by said sperm; and d) determining the percentage of sperm in said aliquot which binds the ligand. In an alternative aspect of this embodiment, step d) can be replaced by the step of assessing the intensity of punctate binding by a detectable label which binds the marker or the ligand either directly or indirectly.

In one aspect of this embodiment the assay encompasses determining the concentration of said marker detected in sperm of said semen sample comprising: a) removing an aliquot from the semen sample; b) contacting the aliquot with a first ligand to said marker; c) detecting binding of said ligand by said sperm; and d) determining the amount of marker expressed by sperm in said aliquot by quantitating the binding of the marker by the ligand; thereby determining the concentration of said marker detected in sperm of said semen sample.

In another embodiment, the ligand is labeled with a detectable label, e.g., a visible label or a radioactive label.

In another embodiment of this assay, detecting the binding of the ligand by the sperm includes detecting label bound directly or indirectly to the sperm, or fragments of the sperm. In one aspect, a sperm fragment is either associated or disassociated from intact sperm.

In another embodiment, detecting the binding of the ligand by the sperm encompasses contacting the sperm in the aliquot with a second ligand which binds to the first ligand.

In another embodiment, the first ligand and/or the second ligand is an antibody. The antibody can be either polyclonal or monoclonal antibodies, and can comprise one or more labels.

In another embodiment of the methods and assays described herein, an indicator of the metabolic status of the sperm is a biochemical marker. The biochemical indicator includes, but is not limited to, one or more of the following biochemical indicators: a cell surface molecule, an enzyme and a receptor.

In one embodiment, the assay is based on the permeability of sperm or fragments thereof, to dyes, in the semen sample and that reflects, or is indicative of, the metabolic status of the sperm. In one aspect of this embodiment the assay encompasses determining the percentage of sperm in the semen sample having the marker that reflects the metabolic status of the sperm encompasses the steps of a) removing an aliquot from the semen sample; b) contacting the aliquot with a dye; c) detecting accumulation of the dye or staining by the sperm or fragments thereof, and d) determining the percentage of sperm or fragments thereof in said aliquot which accumulate dye (or stain), and/or the intensity of staining and/or the quality of the staining (whether uniform or punctate) of the sperm or fragments thereof in said aliquot by the dye. The intensity can be determined by visual observation.

One embodiment described herein encompasses a method of optimizing sperm performance of a semen sample upon insemination of said semen sample, comprising: i) selecting a marker, wherein expression of the marker in the semen sample changes during capacitation; ii) determining the level or location of expression of the marker in the semen sample at a plurality of time points during incubation of said semen sample before insemination of said semen sample; iii) determining a timepoint for preparing said semen sample for use in insemination, wherein said timepoint is based upon a calibration of the marker expression displayed by said semen sample in step (ii) to a kinetic model of biomarker expression during sperm capacitation relative to said performance; iv) preparing said semen sample at said timepoint of step (iii); for use in insemination

Another embodiment entails a method of optimizing sperm performance of a semen sample upon insemination of said semen sample, encompasses the step of i) selecting a marker, wherein expression of the marker in the semen sample changes during capacitation; ii) determining the level or location of expression of the marker in the semen sample at a plurality of time points during incubation of the semen sample before insemination of said semen sample; iii) determining a time point for preparing said semen sample for use in insemination, said time point based upon a calibration of the marker expression displayed by said semen sample in step (ii) to a kinetic model of biomarker expression during sperm capacitation relative to said performance; iv) preparing said semen sample for use in insemination at said timepoint of step (iii); thereby optimizing said performance of said semen sample upon insemination of said semen sample.

Another embodiment entails a method of optimizing sperm performance of a semen sample upon insemination of said semen sample, wherein said semen sample has been exposed to a treatment which modulates the rate of capacitation, encompassing the steps of i) selecting a marker, wherein expression of the marker in the semen sample changes during capacitation; ii) determining the level or location of expression of the marker in the semen sample at a plurality of time points after exposure to said treatment and during incubation of the semen sample before insemination of said semen sample, iii) determining a time point for preparing said semen sample for use in insemination, said time point based upon a calibration of the marker expression displayed by said semen sample in step (ii) to a kinetic model of biomarker expression during sperm capacitation relative to said performance; iv) preparing said semen sample for use in insemination at said timepoint of step (iii); thereby optimizing said performance of said semen sample upon insemination of said semen sample.

The semen sample is mammalian, preferably including, but not limited to human, bovine, equine, canine, feline and murine. The sperm performance is any performance, i.e., a trait or property of interest, including, but not limited to fertility, gender bias, male or female gender bias, or combination thereof. Other performances of interest include the sperm's resistance to processing of said sample for storage/insemination and/or the sperm's resistance to freezing and encapsulation during processing of the semen sample for storage/insemination.

The marker(s) being assayed before insemination of an individual semen sample which is being optimized for an optimum performance according to the methods described herein, can be the same or different from the biomarker(s) used to calibrate the performance of interest. In a preferred embodiment the marker and/or the biomarker is an Fc receptor. An Fc receptor as used herein encompasses a ligand that binds to a region other than the variable domain of an antibody. Accordingly, an Fc receptor as used herein encompasses a ligand that binds to the constant region of an antibody, for example to a constant domain of an antibody. In another embodiment the assay comprises more than one marker. In one embodiment, the calibration means is based upon a kinetic model of biomarker expression during sperm capacitation. The kinetic model of biomarker expression during sperm capacitation is reflected as a measurement over time of an expression pattern of one or more biomarkers, against which performance of interest is correlated. In a preferred embodiment, the biomarker is an Fc receptor.

In one embodiment of the methods described herein, the semen sample is not treated by fixation. In one embodiment of the methods described herein, the semen sample is not exposed to treatment to permeablize the membrane.

In one embodiment of the methods described herein, the first of said plurality of time points is obtained immediately (T₀) after collection of said semen sample. In one embodiment of the methods described herein, subsequent time points of the assay occur at any number of time intervals. The range of the time intervals can be as short as 0.1 minute or less to as long as an hour or more over a period of up to 6 hours or more after T₀. Alternately, the assay points can merge into a continual monitoring over the entire incubation period or parts thereof. In one embodiment of the methods described herein, the marker and the biomarker are identical. In a preferred embodiment, each of the marker and the biomarker is an Fc receptor.

In one embodiment of the methods described herein, the method comprises more than one marker and/or more than one biomarker. In one embodiment of the methods described herein, the kinetic model of biomarker expression during sperm capacitation comprises more than one biomarker. Preferably, the kinetic model of biomarker expression during sperm capacitation comprises an Fc receptor as a biomarker. In an embodiment of the methods described herein, the kinetic model of biomarker expression during sperm capacitation is developed using ejaculates from the same species of said semen sample being optimized for a performance.

In an embodiment of the methods described herein, the preparation of the semen for insemination occurs at 4° C. In another embodiment, the preparation step occurs at room temperature. In another embodiment, the preparation step occurs at a temperature ranging from 0° C. to up to 25° C., 32° C., 37° C., and 42° C. In an embodiment of the methods described herein, the preparation step includes freezing the semen sample. Freezing methods include vitrification processes, in addition to traditional freezing methods. In an embodiment of the methods described herein, the preparation step includes stabilizing said semen sample. Preparation of the semen sample for insemination can be performed using standard methods, including, but not limited to freezing the semen sample with or without additives and/or excipients.

In an embodiment of the methods described herein, where the semen sample has been exposed to a treatment which modulates the rate of capacitation, the treatment accelerates the rate of capacitation. In another embodiment of the methods described herein, where the semen sample has been exposed to a treatment which modulates the rate of capacitation, the treatment decelerates the rate of capacitation. In an embodiment of the methods described herein, where the semen sample has been exposed to a treatment which modulates the rate of capacitation, the treatment comprises exposing the semen sample to a chemical agent which modulates the rate of capacitation. Such a chemical agent includes but preferably is not limited to endocannibinoids, bicarbonate, bovine serum albumin and the non-hydrolyzable analog of cAMP, 8-buteryl cAMP. Additional suitable chemical agents include, but preferably are not limited to, cAMP. caffeine, heparin, bovine serum albumin, glycoproteins, glycans, glycoaminoglycans, sulfated glycans, fucose, cholesterol-loaded cyclodextrins, desmosterol sulfate, kinase inhibitors such as phosphatidyl inositol-3-kinase inhibitor LY294002, phospholipase A2 inhibitor aristolochic acid, Src family kinase inhibitor SU6656, protein phosphatase inhibitor Calyculin A, cyclic GMP-specific phosphodiesterase inhibitor sildenafil, pH-changing buffers such as sodium acetate buffer pH 4, or tris-glycine buffer pH 9, pentoxyfilline, progesterone, ovarian tubal fluid, follicular fluid, seminal plasma from vasectomized or otherwise subfertile males, such plasma being of different ages, and purified components thereof, lipases such as phospholipase A2, glycosidases such as sialadases, proteases such as acrosin, protease inhibitors such as soybean trypsin inhibitor, lectins such as Concanavalin A, ion channel blockers such as tetrodotoxin phenylpropanolamines (cathein, norephedrine), glycolytic pathway intermediates such as pyruvate, the prostatic TRH-related peptide pyroglutamylglutamylprolineamide, fluoride, mood stabilizers such as valproate, topirimate and carbonic anhydrase.

In an embodiment of the methods described herein, where the semen sample has been exposed to a treatment which modulates the rate of capacitation, the treatment comprises exposing the semen sample to a mechanical or environmental stimulus which modulates the rate of capacitation. The mechanical and/or environmental stimulus includes, but preferably is not limited to temperature changes, e.g., raising and/or lowering temperature at specific rates or stepwise increase/decrease, imposed at various times post-ejaculation, mechanical agitation alone, and/or combined with atmospheric changes to induce changes in dissolved gases, use of a mechanical bubbler such as seen on aquarium pumps, to introduce various gaseous agents such as nitric oxide, nitrous oxide, ozone, superoxide, nitrogen gas, carbon dioxide, barometric pressure changes to increase or decrease pressure, alone or in combination with atmospheric changes.

In an embodiment of the methods described herein, where the semen sample has been exposed to a treatment which modulates the rate of capacitation, the treatment comprises exposing the semen sample to an atmospheric condition which modulates the rate of capacitation. The atmospheric condition includes, but preferably is not limited to a CO₂ concentration greater than 5%, and/or an atmospheric condition that comprises one or more of nitric oxide, ozone, argon, and cyanide, or any agent that can be nebulized or otherwise introduce into the atmosphere above a collection.

In an embodiment of the methods described herein, the marker or biomarker is expressed in the extracellular portion of said semen sample and/or is expressed by a sperm cell. In one embodiment, the marker or biomarker is expressed on the cell surface of a sperm cell. In one embodiment, the marker or biomarker is expressed on a membrane of a sperm cell. The membrane includes a cell surface membrane or an outer membrane of a sperm cell. In one embodiment, the marker or biomarker comprises the degree or amount of membrane isotrophy, membrane fluidity, membrane charge, membrane permeability, membrane budding and membrane hydrophobicity. In another embodiment, the marker or biomarker comprises a lipid or a molecule comprising a lipid. A preferred, non limiting embodiment is phosphatidyl serine.

In one embodiment, the marker or biomarker is expressed in an intracellular location of a sperm cell, including, but preferably not limited to intracellular pH, intracellular concentration of HCO₃, intracellular concentration of fragmented DNA and intracellular concentration of Calcium. In an embodiment of the methods described herein, the marker or biomarker is an anionic polysaccharide. The anionic polysaccharide includes, but is preferably not limited to glycosaminoglycans, sulfated glycosaminoglycans and sulfated polylactosaminoglycans. The glycosaminoglycan includes, but is preferably not limited to heparin, fucoidan, and a Lewis antigen.

In an embodiment of the methods described herein, the marker or biomarker binds a dye or displays a differential ability to uptake and/or expel a dye. Nonlimiting examples of dyes include Evans blue, chlorazol Black E, Coomassie Blue and Trypan blue. In an embodiment of the methods described herein, the marker or biomarker comprises a physiologic activity including, but preferably not limited to the frequency of beating of flagella of said sperm cell, the ability of said sperm cell to penetrate mucus, changes in adhesion of the sperm cell, orientation of the sperm cell under external stimuli, hypoosmotic swelling of the sperm cell and the metabolic status of the sperm cell. In an embodiment of the methods described herein, the marker or biomarker comprises oscillatory patterns of dye binding, at increasing and decreasing levels.

In an embodiment of the methods described herein, the marker or biomarker comprises a molecule secreted or expelled by said sperm cell. The secreted or expelled molecule includes, but is preferably not limited to a proenzyme, an agent, a reactive oxygen species, an exosomal vesicle, a dye, and agglutinated product and DNA fragments. The agglutinated product can comprise an antibody, multiple antibodies, primary and secondary antibodies, and/or a conjugated antibody.

In an embodiment of the methods described herein, the marker or biomarker comprises a change in lipid raft structure. In an embodiment of the methods described herein, the marker or biomarker comprises a change in lipid subdomains of the membrane. In an embodiment of the methods described herein, the marker or biomarker comprises a change in lipid subdomains of the membrane. In an embodiment of the methods described herein, the marker or biomarker comprises a change in ion channel permeability, permeability to a dye, tyrosine phosphorylation, kinase activation, protease activation, calcium gradients, mitochondrial function indicators, elaboration of enzymes into the medium, elaboration of specific agents into the medium such as vesicles, free small molecules and products of capacitation reactions.

Membrane lipids and ion channel permeability are measured according to protocols known to one in the art and using commercially available kits, see for example Hug et al. Methods Mol. Biol. 2011; 741:489-509.

Membrane organization including domains and anisotrophy,—can be assessed using art recognized techniques including for example those taught by Balogh et al. (2011) PLoS ONE 6(6): e21182. doi:10.1371/journal.pone. Membrane potential can be measured using assays that are well known in the art and include exposing the cells to dyes and measuring the amount of fluorescence produced as an indicator of membrane potential. Dyes are also used as an indicator of Membrane Potential,—cells are exposed to dyes and the amount of fluorescence produced is an indicator of membrane potential, see for example Waggoner et al., Annual Review of Biophysics and Bioengineering:8: pages 47-68, June 1979). Methods of assaying signaling mechanisms involved in the regulation of sperm capacitation, such as those induced by inhibition of Na(+)/K(+)ATPase activity, are known in the art, see for example, Newton L D et al. Mol Reprod Dev. 2010 February; 77(2):136-48. Methods of assaying membrane phospholipid asymmetry in sperm are known in the art, see for example, the flow cytometric procedure of Gadella, B. M. et al. Development 127, 2407-2420 (2000), in which an increase membrane lipid disorder were detected by merocyanine.

Evoked Targeting

In an embodiment of the methods described herein, the marker or biomarker comprises a change in the response of a semen sample over time to an added agent. The added agent can, for example, evoke expression of or a change in a measurable change in a target molecule or physiology of the semen sample and/or the sperm, andor the environment/medium of the sperm and/or a vesicle shed from the sperm.

Many biomarker targets that have been discovered and employed in the instant invention arise on the cell surface during the course of the assay, as time elapses. However, these do not constitute the entire biomarker target universe. We know the sperm treatment (collection methods and sperm incubation during assay periods) and assay methods augment appearance of the targets we detect (for example, signal intensity changes with temperature, and we use a temperature producing a robust signal). The assays described herein can be extended to additional targets by adding agents to sperm or to the assay to provoke additional measurable changes, with these changes potentially being associated with the presence of or gain of a specific metabolic state—or associated with completion of passage through a specific metabolic state. Changes can involve appearance of specific assay targets, or failure of appearance. Targets can be on or in sperm, in the medium surrounding sperm, or in vesicles shed from sperm, etc. As a nonlimiting example, sperm are placed in an enclosed device and volatiles arising from the collection are sampled and assayed with time. Such evoked changes include, for example, the ability to undergo the acrosome reaction in response to addition of ionophore to the medium. In another aspect of this embodiment, an agent is added that retards attainment of a specific metabolic state—but only in those sperm that are less mature, as opposed to those sperm that are more mature. For example, early stages of capacitation are thought to be reversible, while later stages are not. Thus, sperm in the early stages would respond to decapacitating agents (for example, cholesterol), while sperm in later stages of maturation would be unresponsive due to passing a critical point of no return. One or ordinary skill in the art will recognize that these evoked assay targets are as readily useful as those arising from the invention's standard assay conditions as described herein. In fact the standard assay contains bovine serum albumin, known to play a role in capacitation by being able to bind cholesterol during its offloading from sperm membranes during maturation.

In another embodiment of the methods described herein, comprises the use of agents that arrest the capacitation process at a predetermined stage of capacitation. Use of this embodiment, allows the combination of several cohorts to be combined into a single, more active cohort. Active sperm have a short life. Cohorts of active sperm must therefore mature sequentially in order to provide a steady supply. This means only a small percentage of sperm are capable of fertilization at a given time. This can be problematic in cases of poor fertility. In such cases, it would be better to have many sperm mature at once, and time the egg to their readiness using ART (assisted reproductive technology). Agents and conditions present near the time of ovulation are known to activate sperm. Application of these agents to create a larger group of mature sperm is possible. Appearance and status of this group of sperm can be detected by the instant invention, and the sperm can be stabilized in a state of readiness to produce the highest possibility, in the current example, of fertility. Thus, by adding an agent(s) to sperm designed to stop the maturation process at a specific stage, sequential cohorts of sperm mature to that specific state but not beyond. Release of the blockage would then result in maturation of a large cohort of synchronized sperm. Due to maturation changes being so rapid, an assay is required to determine sperm status and therefore timing of freezing or insemination to produce the desired improvements in sperm performance.

In an embodiment of the methods described herein, the cell whose performance is the subject of optimization is not limited to sperm cells. The methods described herein can be applied to any cell undergoing a metabolic change, including hybridoma cells, stem cells, embryonic stem cells, microbes used in preparation of fermented foods or materials, or cells for transplant, as nonlimiting examples. With respect to methods described herein comprising hybridoma cells, the performance of interest may comprise for example, optimum antibody secretion and/or cell viability and/or resistance to subsequent processing. In one embodiment a method of optimizing performance of interest of hybridoma cells in a culture comprising said hybridoma cells, comprises: i) selecting a marker, wherein expression of the marker in said culture changes over time; ii) determining the level and/or location of expression of the marker in the culture at a plurality of time points before harvesting of said culture; iii) determining a time point for harvesting the culture based upon a calibration of the marker expression displayed by the culture in step (ii) to a kinetic model of biomarker expression during hybridoma cell culture relative to said performance of interest; iv) processing the culture at said time point of step (iii); thereby optimizing said performance of interest of said hybridoma cells upon harvesting of said culture.

With respect to methods described herein comprising stem cells, the performance of interest may comprise for example, optimum developmental stage and/or cell viability and/or resistance to subsequent processing. In one embodiment, a method of optimizing performance of interest of stem cells in a culture comprising said embryonic cells, comprises: i) selecting a marker, wherein expression of the marker in said culture changes over time; ii) determining the level and/or location of expression of the marker in the culture at a plurality of time points before harvesting of said culture; iii) determining a time point for harvesting said culture based upon a calibration of the marker expression displayed by said culture in step (ii) to a kinetic model of biomarker expression during stem cell culture relative to said performance of interest; iv) processing the culture at said time point of step (iii); thereby optimizing said performance of interest of said stem cells upon harvesting of said culture.

With respect to methods described herein comprising cells to be transplanted into an individual, the performance of interest may comprise, for example, cell viability and/or resistance to subsequent processing and/or reduced alloantigen expression.

In one embodiment, a method of optimizing performance of interest of cells for transplant in a culture comprising the cells for transplant, comprises: i) selecting a marker, wherein expression of the marker in the culture changes over time; ii) determining the level and/or location of expression of the marker in the culture at a plurality of time points before harvesting of the culture; iii) determining a time point for harvesting the culture based upon a calibration of the marker expression displayed by the culture in step (ii) to a kinetic model of biomarker expression during culture relative to said performance of interest; iv) processing the culture at said time point of step (iii); thereby optimizing the performance of interest of the transplant cells upon harvesting of said culture.

DEFINITIONS

In accordance with this detailed description, the following abbreviations and definitions apply. It must be noted that as used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody” includes a plurality of such antibodies, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th) Ed, John Wiley & Sons, Inc. See Harlow and Lane, Antibodies: A Laboratory Manual, Cold Springs Harbor Publications, New York, (1988) which are incorporated herein by reference) and chemical methods. Unless otherwise stated, all ranges described herein are inclusive of the specific endpoints. The following terms are provided below.

As used herein, the term “desired trait” or “performance” with respect to sperm includes, but is preferably not limited to, a physiological characteristic of the sperm. The desired trait can be expressed before fertilization, e.g., the trait of being in a physiologic state capable of fertilization. Alternatively, the desired trait can be expressed after fertilization or both before and after fertilization, e.g., gender of fetus or newborn. The desired trait or performance also includes the reisistance or ability of the sperm to withstand preparation of the semen sample or portion thereof, for insemination after the assay methods of the invention. Accordingly, a performance can include the ability of a semen sample to withstand a process used to prepare the semen sample or portion thereof for insemination. Processes for preparing a semen sample for insemination include freezing or vitrification or dehydrating the semen sample to be inseminated. Processes for preparing a semen sample for insemination also include the addition of protective agents to the semen sample to be inseminated, and/or encapsulation of the semen sample or portion thereof. The timing of the expression of the desired trait or performace relative to time zero (the time of the collection of the ejeaculate containing the semen sample when assayed immediately) varies between semen samples, as illustrated in FIG. 11.

As used herein, the term “semen sample” includes any semen sample collected from an ejaculate of any mammal, including, but preferably not limited to, human, cattle, goats, sheep, buffalo, swine, horses, cats, dogs, rat, mouse, rabbits, hamsters and endangered species of mammals. A semen sample can be obtained from both first and second ejaculates, and electro ejaculated collections, for example from bull studs. The semen sample is frozen, and later incubated according to the assays as detailed below. Before incubation and/or before undergoing a biomarker assay, optionally the semen sample or a portion thereof, is washed, and/or diluted, optionally with a buffered saline solution. Preferably the sample is incubated at a constant temperature immediately after collection. In a preferred embodiment, the semen sample undergoes a gentle temperature gradient from approximately up to 40° C. immediately after obtaining an ejaculate, to approximately 12° C., or below, or an alternate temperature, until the semen sample is processed for insemination. The semen sample typically undergoes at least a first assay for marker expression which is above endpoint temperature.

As used herein, the term “metabolic status” or “metabolic state” or “metabolic stage” is a physiological state of the sperm with respect to specific biochemical and biophysical properties at a specific time point during the incubation period of the sperm sample. Typically, the physiology of sperm changes over time with age, such as the physiological change in fertility of sperm, as illustrated in FIG. 12. Thus, changes in physiology or metabolic state of sperm in a semen sample can include expression of a desired trait, e.g., fertility. Additionally, changes in physiology or metabolic state of sperm in a semen sample can be reflected by expression of one or more markers. In one embodiment of the methods disclosed herein, the expression of one or more markers occurs earlier than the expression of the desired trait. Further, Applicant has discovered that the expression of such markers by sperm in the semen sample occurs at a predetermined time interval, i.e., a predetermined number of minutes or hours, before the expression of the desired trait.

Though the metabolic changes associated with sperm activation is accelerated in Y bearing sperm relative to X bearing sperm, the exact timing of the occurrence of these changes may vary with each individual semen sample. However, the interval between an earlier metabolic state reflected by expression of a marker and the later expression of the desired trait, e.g., fertility, is constant (other conditions, e.g. temperature, being constant). This defined time interval is used in the methods described herein to determine when each individual sample will express the desired trait i.e., performance, e.g., fertility, by monitoring expression of one or more markers. Thus, the time for processing semen into straws can be determined with an increased probability of obtaining the desired trait or performance.

Thus, the phrase “a marker indicative of a metabolic stage of sperm” refers to a measurable attribute of a sperm, including but not limited to a physiological, structural, functional, biochemical and/or electrochemical attribute which reflects the metabolic status of the sperm at a particular time point. The term “marker” and “biomarker” which are used interchangeably herein, includes, but is preferably not limited to, a structural, physical, electrophysical, physiological or biochemical attribute of a semen sample, including the seminal fluid, sperm, and/or fragments thereof, including, for example, the nonlimiting examples of acrosome length, sperm morphology (ruffling) of the sperm, expression of a cell surface molecule on the sperm, electrostatic charge of sperm, and permeability by sperm to a molecule, such as, for example, but not limited to, a dye.

The term “marker” and “biomarker” further includes, but is not limited to, a ligand, a lectin, an enzyme and a receptor, which is expressed on the surface of the sperm, or internally, or both, and/or in the seminal fluid. In some embodiments, the marker is a morphological change in an acrosome which can be viewed, for instance, using bright field microscopy. With respect to acrosome morphology, over time the surface of the acrosome's membrane appears increasingly ruffled. In some embodiments a marker can be cryptic at some stages of metabolism, and not detected. Though the terms “biomarker” and “marker” are used interchangeably, their recitation can be used to distinguish comparative populations, for example expression by the sample being assayed vs. the sample(s) used as a control or for calibration.

Thus, calibration ties the biomarker expression levels of a semen sample to performance of a desired performance or trait of the semen sample during or after insemination, e.g., fertility and/or gender bias, and also including resistance to processing for insemination and/or storage preceding insemination. Biomarker expression levels of a semen sample can be assayed over time for the percentage sperm which are positive for the biomarker for example. In another embodiment, biomarker expression levels of a semen sample can be assayed over time for the greatest change in expression of a biomarker. In another embodiment, biomarker expression levels of a semen sample can be assayed over time with respect to oscillatory patterns. Thus even if a maximum expression level is not observed, the biomarker expression is analyzed with respect to the number of oscillatory cycles. These cycles include pattern changes of populations as measured, for example, by cytometry, etc.

The calibration correlates the performance of interest of sperm obtained at a time point relative to biomarker expression over multiple timepoints (e.g., period of maximum level, or maximum change over time, or oscillation pattern) forming the kinetic model. The calibration can be against the kinetic model of any mammalian sperm. For example, in choosing the optimum time point to prepare a particular human semen sample for insemination, the calibration can be performed in the context of a kinetic model of sperm performance in which the sperm is not necessarily limited to human sperm. The kinetic model is useful in calibrating the extent to which an individual semen sample is undergoing capacitation at reduced or increased rate relative to the typical rate.

Thus the kinetic model provides a typical rate and timecourse of capacitation as evaluated through the use of one or several biomarkers associated with capacitation in semen. The kinetic model also correlates a performance of interest to a timepoint for preparing an individual semen sample for insemination based on the pattern of biomarker(s) expression, such that the individul semen sample will achieve the optimal performance of interest. The use of this kinetic model can be used to calibrate the expression pattern of one or more markers by an individual semen sample over time, to accommodate a reduced or accelerated or an otherwise altered progression of capacitation in an individual semen sample.

Preferably, in the present methods, the optimal expression of the desired sperm trait/performance occurs consistently after a constant number of minutes or hours has elapsed from the time that the sperm achieved a specific metabolic state, as illustrated in FIG. 13. Whether the sperm has achieved the specific metabolic state can be assessed by expression of one or more markers by the sperm.

As used herein, the term “antibody,” includes, but is not limited to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, an IgG antibody, an IgM antibody, or a portion thereof, which specifically bind and recognize an analyte, antigen or antibody. An antibody or fragment thereof comprises an antibody or fragemtn thereof which is isolated froma natural source, for example an animal, mammal, mouse or human. Alternatively an antibody or antibody fragment is produced using synthtic processes, including but not limited to recombinant methods, and chemical syntheisis. “Antibody” also includes, but is not limited to, a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, which specifically binds and recognizes the antigen-specific binding region (idiotype) of antibodies produced by a host in response to exposure to the analyte. In one embodiment of the methods described herein, an antibody binds the sperm or fragments thereof, or a primary antibody, through a site on the antibody other than its paratope. In another embodiment, the antibody binds the sperm or fragments thereof, or a primary antibody through its paratope. In another embodiment of the methods described herein, an antibody binds the sperm or fragments thereof, or a primary antibody, both through its paratope and through a site on the antibody other than its paratope. Reactive molecules similar to antibodies, but derived from phage display libraries, are also included.

As used herein, the term “antibody,” encompasses polyclonal and monoclonal antibody preparations, as well as preparations including monoclonal antibodies, polyclonal antibodies, hybrid antibodies, altered antibodies, F(ab′)₂ fragments, F(ab) fragments, F_(v) fragments, single domain antibodies, chimeric antibodies, humanized antibodies, dual specific antibodies, bifunctional antibodies, single chain antibodies, and the like, and functional fragments and multimers thereof, which retain specificity for an analyte or antigen. For example, an antibody can include variable regions, or fragments of variable regions, and multimers thereof, which retain specificity for an analyte or antigen. See, e.g., Paul, Fundamental Immunology, 3rd Ed., 1993, Raven Press, New York, for antibody structure and terminology. Alternatively, the term “antibody” comprises a fragment thereof containing the constant region, in particular the Fc region. The antibody or portion thereof, may be derived from any mammalian species, e.g., from a mouse, goat, sheep, rat, human, rabbit, or cow antibody. An antibody or fragments thereof, may be produced synthetically by methods known in the art, including modification of whole antibodies or synthesis using recombinant DNA methodologies, including using phage display libraries.

As used herein, the phrase “binds to” refers to an antibody, reagent or binding moiety's binding of a ligand with a binding affinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater, more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art (for example, by Scatchard analysis). A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an analyte. See Harlow and Lane, Antibodies: A Laboratory Manual, Cold Springs Harbor Publications, New York, (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically a binding reaction will be at least twice background signal to noise and more typically more than 10 to 100 times greater than background.

An antibody may be used to detect a marker or biomarker of sperm and or of the semen sample, both through the antibody's paratope and/or through a site on the antibody other than its paratope. In a preferred embodiment, the antibody is not limited to detecting a marker or biomarker through a primary antibody's antigen binding site. though not being bound to theory, subsequent binding or association of the primary antibody to a secondary antibody may result in aggregates which are bound to, or asociate with, the interior, exterior and/or membrane portion of a sperm. The change in detection of antibody aggregates over time is a preferred marker/biomarker of the assays of the invention described herein.

As used herein, the term “capacitation” encompasses the physiological changes the spermatozoa must undergo in the female tract or in vitro before being capable of penetrating the ovum. (Saunders Comprehensive Veterinary Dictionary, 3 ed. © 2007 Elsevier, Inc. Capacitation is distinct pathway from pseudocapacitation, but does not lead to the complete acrosome reaction. A biomarker or marker of the assays disclosed herein excudes the use of the complete acrosome reaction as a marker or biomarker.

As used herein, the term “label” includes a detectable indicator, including but not limited to labels which are soluble or particulate, metallic, organic, or inorganic, and may include radiolabels (such as, e.g., ¹⁴C, ³H, ³²P) enzymatic labels (e.g., horseradish peroxidase, galactosidase, and other enzyme conjugates), spectral labels such as green fluorescent protein, fluorescent dyes (e.g., fluorescein and its derivatives, e.g., fluorescein isothiocyanate (FITC), Alexa Fluor® 488 Dye, which is a green-fluorescent dyes conjugate with nearly identical spectral properties and quantum yield as fluorescein isothiocyanate, rhodamine, Yo-Pro, a carbocyanine nucleic acid stain sold by Invitrogen, catalog Product V13243, the green-fluorescent YO-PRO®-1), chemiluminescent compounds (e.g., luciferin and luminol), spectral colorimetric labels such as colloidal gold, or carbon particles, or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads, as well as dyes, including the cell-permeant pH indicator, carboxy SNARF®-1, an acetoxymethyl ester, acetate which has a pKa of ˜7.5 after de-esterification and is sold by Invitrogen, as catalog #PPLM63-C1270. Where necessary or desirable, particle labels can be colored, e.g., by applying dye to particles.

This, the label can be detected using colorimetric platforms with enzyme-produced color like in ELISA type tests. Luminometers can also be used.

As used herein, the term “colored particle label” includes, but is not limited to colored latex (polystyrene) particles, metallic (e.g. gold) sols, non-metallic elemental (e.g. Selenium, carbon) sols and dye sols. In one embodiment, a colored particle label is a colored particle that further comprises a member of a conjugate pair. Examples of colored particles that may be used include, but are not limited to, organic polymer latex particles, such as polystyrene latex beads, colloidal gold particles, colloidal sulphur particles, colloidal selenium particles, colloidal barium sulfate particles, colloidal iron sulfate particles, metal iodate particles, silver halide particles, silica particles, colloidal metal (hydrous) oxide particles, colloidal metal sulfide particles, carbon black particles, colloidal lead selenide particles, colloidal cadmium selenide particles, colloidal metal phosphate particles, colloidal metal ferrite particles, any of the above-mentioned colloidal particles coated with organic or inorganic layers, protein or peptide molecules, or liposomes. For example, Quantum dots sold by Invitrogen, is a label encompassed herein.

As used herein, the term “decreased expression” with respect to a marker, refers to a decrease in expression (including a decrease in accessibility or an increase in crypticity) of a marker or given measurable activity (e.g., binding activity, membrane permeability, electrostatic charge) by at least 5% relative to a reference. Such decreased expression is down regulated by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, up to and including 100%, i.e., complete absence of the given activity. Decreased expression of a marker can be measured as described in the working examples herein. The term “increased expression” refers to an increase in expression of a marker or given measurable activity (e.g., binding activity, membrane permeability, electrostatic charge) by at least 5% relative to a reference, for example, at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%. An increased expression of a marker can be measured as described in the working examples herein.

As used herein, the term “gender bias” with respect to the sperm in a semen sample, refers to a sample having a greater proportion of active, fertile sperm carrying an X chromosome (female gender bias) or a Y chromosome (male gender bias) relative to the proportion of active, fertile sperm carrying an Y chromosome or an X chromosome, respectively. A gender bias in a semen sample, for example in a semen sample processed in accordance with the methods disclosed herein, may be reflected in the number of relative proportions of male and female offspring generated from an individual semen sample. In preferred embodiments, the gender bias provided by these methods exceeds a gender bias which may be typical of a particular species or individual animal. The methods described herein take advantage of a natural tendency for Y bearing sperm in a semen sample to mature at a faster rate than X bearing sperm, by providing for the identification of when this differential maturation reflects a significant gender bias among sperm with high fertility rates. A particular semen sample will have a gender bias despite the variant rates of sperm maturation found in each collection, but each sample will have the capability of this bias at different times post collection of the ejaculate from the animal.

As used herein, the term “fertility” with respect to sperm in a semen sample, refers to the ability of the sperm to fertilize an egg and create a viable fetus and live-born animal. This ability changes as the sperm age, and changes differentially with respect to whether the sperm is carrying an X chromosome or a Y chromosome.

As used herein, the term “capacitation” refers to the development pathway sperm cells naturally under go to become fertile after ejaculation. The progress of sperm along the pathway to fertility can be monitored by assaying the expression of the markers and biomarkers described herein. In embodiments herein, the rate of capacition may be modulated with the use of externally added agents. In one embodiment of the invention, the agents are added to the assay, and the biomarkers of the invention are assessed. In another embodiment, the agents are added to the semen sample itself at the time of processing for use in insemination and or storage. Preferably, the term “capacitation” does not include the process of pseudo capacitation, which has been reported at lowered temperatures.

As used herein, the term “punctate staining” means a distribution of detectable label in the form of distinct spots or points as opposed to a uniform staining across the surface of a sperm or fragments thereof. Quantitative analysis of punctate staining is known in the art, as described for example by Maxim Mokin and Joyce Keifer in “Quantitative analysis of immunofluorescent punctate staining of synaptically localized proteins using confocal microscopy and stereology”, Journal of Neuroscience Methods, Volume 157, Issue 2, 30 Oct. 2006, Pages 218-224.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Assays used as research tools to evaluate the changes in sperm capacitation that occur with time

FIG. 2. Description of Technology

FIG. 3. Fluorescence Assay

FIG. 4. Comparison of Semen Processing by Standard Methods in the Art and Applicant's Methods. Particularly in processing of human sperm, wash steps are added prior to dilution and freezing, but are not shown here. Alternatively, prepared sperm are sometimes used fresh without freezing.

FIG. 5. Process Control Assays Sperm Cell Assays Reveal High Variability. (A) assay of collections on two different days from a Canadian Holstein bull. (B) assay of collections on two different days from a Friesian bull. Y axes are percentage of cells which are positive for the marker. X axes are time of neat semen incubation.

FIG. 6. Improvement of Fertility using Applicant's methods described herein

FIG. 7. Field Data showing increased fertility using Applicant's methods described herein

FIG. 8. Field Data showing increased gender bias using Applicant's methods described herein

FIG. 9. Field Data showing increased gender bias using Applicant's methods described herein

FIG. 10. Fertility and Gender Bias Increases In Working Dairy Herds (A) bull RDU collection cohorts E (assay-based) and F (fixed time); (B) RDU collection cohorts M (fixed time) and N (assay-based).

FIG. 11. Y Sperm Gains—and loses—the Ability to Fertilize before X does

FIG. 12 Assay shows that metabolic status of sperm changes with age.

FIG. 13. Process Control uses assay of metabolic status to determine timing of desired trait, e.g. fertility and/or gender bias

FIG. 14, Assays show Jump points vary with collection. FIG. 14A. uses semen collected from the bull Barcardi. FIG. 14B. uses semen collected from the bull RDU.

FIG. 15 Applicant's Model of Process Control illustrates that the gender bias is different for every ejaculate

FIG. 16 High-through put Cytometry facilitates marker assays described herein.

FIG. 17A illustrates the art recognized process of maturation/capacitation of freshly ejaculated sperm (Rausch and Kortleever, 2011).

FIG. 17B illustrates the art recognized coordination of sperm capacitation with egg binding and fertilization (from Flesch and Gadella, 2000).

FIG. 17C illustrates additional art recognized details of fertilization (from Ikawa et al., 2010)

FIG. 18A illustrates art recognized details of fast events of capacitation, Salicioni et al., 2007.

FIG. 18B illustrates art recognized details of slow eventsof capacitation, Salicioni et al., 2007.

FIG. 19. Biomarker profiles of different biomarkers and human semen samples, illustrating human donor sperm kinetic against the biomarker assay and other biomarkers run simultaneously on the same ejaculate, with ejaculates from different donors.

FIGS. 20A and 20B: Application of the antibody-based Fc receptor biomarker to optimize sperm fertility in humans or gender bias in cattle, shown for human Assisted Reproductive Technology (ART)(FIG. 4A), and for frozen doses of cattle semen used on dairy farms (FIG. 4B). 10 ul of human semen are assayed, and 5 ul of cattle semen.

FIG. 21. Acquisition of fertilizing ability by sperm involves profound real-time changes in the acrosome that begin post-ejaculation and continue for hours. Acrosomal changes begin at left.

FIG. 22. Comparison of biomarker sperm status by scanning electron and fluorescent microscopy.

FIG. 23. A. Real-time assays were used before inseminations to optimize bull sperm performance. Top panel, examples of profiles from 4 different bulls, showing arrows corresponding to the different times post-collection at which each ejaculate reached a maximum percentage of biomarker positive cells. Bottom Panel, comparison of biomarker kinetics of antibody and dye assays on an ejaculate from one human donor. Assays were run as described in the OptiFert SOP, with the following change for the dye assay: Primary antibody, designated as Red 2, and secondary antibody, designated as Blue 3 (see SOP for detailed disclosure of antibody types and catalog numbers), were omitted.

B. Processing was carried out by use of the veterinary SOP, except that scoring was by microscope, not cytometer. Scoring by microscopy involved counting, by brighfield or fluorescence as required, the number of biomarker positive and negative cells, and calculating the % positive cells. Conventional semen was processed according to the procedures in use at that dairy bull stud. Inseminations were the standard type used in bovine artificial insemination with deposition of semen in the uterus, just beyond the cervix. Calf sex was determined at birth, with a 10-day calving interval to enable tracing of calf to intended sire and to eliminate service to a different sire.

C. Production of statistically significant (p=0.10) increased fertility by the biomarker-half of 5 split ejaculates compared to control ejaculate half that was also incubated, but not according to biomarker assay timing, on dairy farms. Control incubation treatment involved dilution and freezing of the ejaculate 1 hour before or 1 hour after the optimal time indicated by the biomarker assay

D. Statistically significant (p=0.03) increased fertility of biomarker ejaculates compared to conventional ejaculates from the same bull, on dairy farms. Semen was processed as described in this figure.

FIGS. 24A and 24B. Timing of multi-point assays to optimize sperm performance.

FIG. 25. Biomarker kinetics, of different markers. A Antibody biomarker was run in comparison to an assay of CD46. B. Assay of soybean trypsin inhibitor (SBTI) C. Assay of motility was determined by evaluation of the number of sperm capable of entering the upper region of a swim up tube, relative to total number of sperm applied to the swim up test, using methods of swim up well-established in the art (see, for example, Paasch et al., 2007). D Evaluation of acrosome-reacted sperm was performed by a trained observer using microscopy. E. Dye binding, reproduced from earlier figure. F. Appearance of biomarker-positive vesicles was determined by a trained observer using fluorescence microscopy.

FIGS. 26A-26D. Photomicrographs of biomarker labeling. (FIG. 26A) Fertility-associated antigen (Sprott et al., 2000). (FIG. 26B) Antibody biomarker, hours-old collection (Wrenzycki and Cohen, unpublished). (FIG. 26C) soybean trypsin inhibitor (Harper et al., 2008). (FIG. 26D) CD46 (Harper et al., 2008).

FIG. 27. A temporal map of biomarker expression during the maturation of a cohort of sperm.

FIG. 28. Association of solubilized zona-binding proteins with the apical ridge of sperm (from Burkin and Miller, 2000).

FIG. 29. Flow Diagram Showing Semen Processing in the Presence and Absence of Process Control by Assay

DETAILED DESCRIPTION

Good fertility and replacement female animals are essential to economic health in the dairy industry. The need for female animals remains unmet by gender bias technologies, because they reduce fertility.

A way now exists to address this great market need, as proven by market adoption of the instant invention, with its novel conceptual basis.

Bias in gender ratios of births occurs naturally in response to environmental conditions in mammals. Therefore, biological mechanisms exist to create this bias. The metabolic status of sperm cells at and after insemination is one determinant of gender bias, among others. One could thus theorize that gender bias could be altered in a controlled fashion, if only one could invent a way to evaluate sperm metabolically in real time, while sperm are being collected from dairy bulls and processed by the dairy bull industry into doses to be sold to dairy cow farmers for artificial insemination of cows and heifers (virgin cows).

Such measurement would then become part of the process control steps used to prepare semen doses to service cows. This has analogy in other industries, where one measures specific parameters during manufacturing steps to insure that the product will function well. Currently with sperm, during processing into doses for cows, one measures parameters such as motility and cell number. The instant invention is an assay used to measure attributes of semen in real time during processing that will in the future relate to fertility and/or to gender bias produced by this semen after insemination of cows.

It is well documented in the field that sperm at the time of collection is dormant, and that as it undergoes metabolic changes enabling the sperm to become activated over time. After reaching maximum activation, the sperm continue with their aging process, becoming less fertile, and eventually degrading.

Lechniak et al. (2003, Reprod. Dom. Anim. 38:224-227); incorporated herein by reference in its entirety) describes a study to determine whether or not sperm pre-incubation prior to fertilization in vitro (IVF) influences the sex ratio among blastocysts. The authors reported that when comparisons between groups were made and the actual sex ratios taken into consideration, there were significantly more female-hatched blastocysts among the 24-hour group than among those of either the O— or 6-hour pre-incubation groups.

The time course of activation of sperm from its dormant state at collection varies not only among individuals within the same species, it also varies between ejaculates obtained from the same individual animal. See FIG. 11. This large degree of variability in the time course of sperm activation led one of skill in the art to conclude that characterizing processes of sperm or obtaining a semen sample with physiology characteristic of a certain time in development such as their fertility or gender bias, was not a process which could be achieved reliably based solely on the time interval after collection of the semen sample.

Thus, described herein are methods that allow one to more accurately determine the time point after collection of the semen sample in which a desired trait is or will be associated with of the sperm relative to an earlier distinct metabolic state. The earlier distinct metabolic state need not have any relevance to the desired trait, other than it occurs at a defined number of minutes or hours before the time point during which the sperm in the semen sample have the desired trait.

Though the metabolic changes associated with sperm activation is accelerated in Y bearing sperm relative to X bearing sperm, the exact timing of the occurrence of these changes varies by up to and including one hour, or by as much as several hours, including up to and including one, two, three, four, five, six, seven, eight, nine, ten, eleven, and twelve hours, or more, with each individual semen sample. However, Applicant has found that the time interval between an earlier metabolic state reflected by expression of a marker and the later expression of the desired trait, e.g., fertility, is constant among individual semen samples collected from various individuals, and/or the same individuals, incubated under the same conditions. This defined time interval is used in the real time methods described herein to determine when each individual sample will express the desired trait, e.g., fertility, by monitoring expression of one or more markers. The time at which the semen sample has the desired traits and can be processed, for example prepared for artificial insemination, for example, can be determined by applying the time interval to the time at which the sperm in the semen sample express a marker at a specific level.

Metabolic state predicts field trait. The concept extends biomarker applciation to process control, specifically, for a group of cells that move from dormancy to activity then to senescence in ways that affect their field performance and can now be measured. Preferably, the defined metabolic state is easily identified by monitoring for the co-expression of a specified marker(s), or for a specific level of expression of a specified marker(s). Once it is determined the time at which the sperm in the semen sample achieved this metabolic state, (also called a jump point), one can determine the later time point when sperm in the semen sample will have the desired trait, by applying the predetermined defined time interval between expression of the label and the desired trait. Thus the jump point is the time point from which one calculates when the desired trait will be expressed in the collected semen sample, by adding the time defined in the predetermined time interval. See FIGS. 13 and 29.

For example, FIG. 11 reflects the prior art's teaching that sperm carrying a Y chromosome attain—and then loose—their peak ability for fertilization earlier than do sperm carrying an X chromosome. Attainment of a particular metabolic state requires passage of time—an amount of time that differs for every ejaculate. In contrast, Applicant notes that the time betweeen attainment of a specific metabolic state and attainment of specific field traits related to that state can be calibrated according to a constant curve generated across ejaculates. Therefore, according to Applicant's working model, by evaluating the timing of metabolic states by assay, and correlating this timing to sperm behavior, it is possible to predict and obtain desired field traits/performance. This is achieved by using the assay to dictate the timing of sperm processing—that is when sperm are diluted and aliquoted into doses, for final processing steps that precede insemination, including artificial insemination.

Since, in accord with the present invention, the time between of the occurrence of an earlier distinct metabolic state of the sperm in the sample (i.e., the “jump point”), and the peak ability for fertilization by sperm carrying a X chromosome, is constant between samples incubated or held under the same conditions, one can determine when any semen sample similarly incubated has, e.g., an increased female gender bias by measuring the time indicated by the graph after the jump point. Processing the semen sample, e.g., for artificial insemination (AI), at this later time point after the jump point, will result in a sample with an increased female gender bias. Similarly, since the time between the jump point and the peak ability for fertilization sperm carrying an X chromosome is constant between samples, one can determine when any semen sample similarly incubated has an increased female gender bias by measuring the time indicated by the graph between the jump point and the peak fertility of male sperm.

Method of Obtaining a Graph Defining the Relationship Between the Jump Point and the Later Time Point when the Sperm in the Incubated Semen Sample Express the Desired Trait/Performance

Described herein are methods to establish the constant, predetermined defined time interval between expression of a marker and a desired trait or performance. Because, as Applicant has discovered, this interval is constant between individual samples incubated or held under the same conditions, the time interval can be established for use in any sample of the same species/strain of animal, based on the establishment of the time interval in a single semen sample. Thus this interval can be applied to individual semen samples regardless of their individual variations in how quickly the sperm in each semen sample begin sperm activation.

A graph such as that illustrated in FIG. 13, defining the relationship between the jump point and the later time point when the sperm in the incubated semen sample express the desired trait or performance, can be obtained by monitoring a change in the metabolic status of sperm in a semen sample during the incubation period after its collection. The semen sample is collected and incubated, and the metabolic status is monitored during the incubation period, encompassing in one embodiment, the following steps of:

-   -   i) determining the change in percentage of the sperm displaying         a marker/indicator of the metabolic status over time during the         incubation or the condition under which the sperm is being held;     -   ii) determining when the sperm in the semen sample display         significant fertility and/or a significant a significant gender         bias and/or a desired trait,     -   iii) selecting a time point (jump point) which occurs before the         sperm display a desired trait, and     -   iv) determining the percentage of sperm in said sample which         display said marker/indicator at the selected time point (jump         point); thereby determining when the jump point occurs for each         marker.

This method allows one to determine the constant time interval, between when a desired trait of the sperm in an individual semen sample occurs relative to an earlier discrete metabolic state of the sperm in the semen sample, a metabolic state that can easily be detected by expression of a marker of any measurable type.

Once the relationship between the jump point and the time interval in which the sperm have a desired trait/performance is determined for each species or each donor, the relationship can be used to determine when the semen sample is most likely to contain sperm having a desired trait, so that the semen sample can be further processed. That is, the incubation of the semen sample can be stopped for further processing, or for storage, etc., at the predetermined time after the jump point.

Using a collection method in which the relationship between the jump point of expression of a specified marker indicative of sperm metabolism, and the later time interval in which the sperm have a desired trait has been determined, allows one to more reliably use a time based assay for overcome the high variability of semen metabolic rates across ejaculates (collections) used in artificial insemination (AI) thereby obviating a source of significant problems. Such a method provides for the ability to monitor the biological processes of sperm in a semen sample during real time. This real time monitoring provides a means to tailor semen processing to each individual collection, so that the semen sample is processed when the desired trait is prevalent, and/or optionally differentially expressed, as discussed, for example, in the next section.

Obtaining the Sample with the Desired Trait/Performance

Once the constant, defined time interval between the jump point (expression by a particular marker(s) reflecting a distinct homeostatic state) and the later time point when the sperm in the incubated semen sample express the desired trait has been established, as described above, the optimum time point for processing the sperm in an individual semen sample that have a desired trait, such as a gender bias, can be determined by a simple assay. The assay involves monitoring the one or more markers to determine when the jump point occurs in the individual semen sample of interest.

Specifically, the optimum time point for processing the sperm in the particular semen sample that have a desired trait, is based on i) the individual timing of the jump point in the particular sample, and ii) the constant, predetermined time interval between the jump point of expression by a particular marker and the later time point when the sperm in the incubated semen sample express the desired trait.

Thus, in one embodiment, aliquots of the sample can be removed from the semen sample of interest at various time points and assayed for expression of the specific marker(s) so that the timing of the jump point for the semen sample of interest can be established. The optimum time point for processing the sperm in the particular semen sample is then determined by simply waiting the number of minutes/hours specified by the pre-determined time interval between the jump point and the later time point when the sperm in the semen sample of interest express the desired trait/performance.

The monitoring encompasses the following steps using the obtained semen sample of:

-   -   i) determining the percentage of sperm in the semen sample         having a marker/indicator of its metabolic status during         incubation post collection at various time points; and     -   ii) determining the time point (jump point) at which a specified         percentage of sperm (or a specified change in the percentage of         sperm) in the semen sample have the marker/indicators; where the         jump point precedes the interval during which the semen have the         desired trait, such as gender bias and/or fertility, by a         defined amount of time as described above.

The defined amount of time can be determined by the first method described in this section. Thus, if it is known, for example, that the optimum fertility occurs two hours after the sperm in the sample have attained a specific metabolic status, then one can process the semen sample two hours after the sperm has reached the specific metabolic status (i.e., jump point).

As discussed above, the time at which the sperm attains the specific metabolic status varies from sample to sample, but can be detected by assaying for a marker which reflects the specific metabolic status. Aliquots can be taken from the incubated semen sample at regular intervals beginning at the time at which the semen sample is first collected. In one embodiment aliquots are taken every minute, 2 minutes, 10 minutes, every 15 minutes, every 20 minutes, every 30 minutes or every 60 minutes. In another embodiment samples are taken at 5 minute intervals. In yet another embodiment aliquots are taken at hour intervals. The interval will depend on conditions of incubation and experience with a particular donor. In still another embodiment only a single aliquot is taken and used. In some embodiments the sampling times are adjusted based on the change detected. Here, sampling times in collections that are rapidly changing are shortened while sampling times in collections that are changing slowly are lengthened. Sampling times within a single collection can also be varied. They can be lengthened for those times during incubation of a semen sample where metabolic change is slow, and shortened for those times during incubation of a semen sample where metabolic change is fast.

ADVANTAGES

Clearly, the full advantage of increased fertility and/or sex selection for farm economics and genetic improvement of the herd base, or for reduction of human disease, has been unreachable due to the lack of an effective fertility and/or gender bias technique suitable for on-site use (e.g., on-farm and in clinics). What is needed is a method that preserves fertility and/or generates a moderate sex bias when used with standard on-farm and in-doctor's office methods of artificial insemination, and eliminates exposure of sperm to mutagens and damaging conditions. An ability to monitor sperm biological processes as they occur during semen processing provides a way to tailor semen processing to each individual collection of semen, thereby optimizing sperm quality and maximizing fertility and/or creating bias. The present assay provides a solution which makes it possible to increase fertility and/or skew the sex ratio of births in methods that is applicable on-site, while simultaneously eliminating exposure of sperm to deleterious conditions and agents.

The methods described herein are designed to capture these advantages. In addition, because the preferred method simply imposes process control on biological processes common to all mammalian sperm, it is broadly applicable. This is especially relevant for breeding of exotic species, including primates, where fertility maintenance is a key factor and is related to the number and quality of cells used during insemination. It is likewise relevant for on-farm use with cattle, sheep, goats and swine or with champion livestock and animals such as race horses and pedigreed dogs and cats, and exotic or endangered species. The technology also has advantages for human users who suffer from fertility issues or sex-linked diseases, as also described herein.

Markers of Metabolic Status

Optimal sperm quality can be defined on the basis of numerous attributes such as number of viable sperm, sperm motility (both the percentage that are motile and the type of motility exhibited), sperm morphology, acrosomal integrity, etc. It is known, for example, that X-bearing sperm are visibly larger than Y-bearing sperm. It is also known that all sperm go through a series of metabolic changes once ejaculation has occurred and the sperm is mixed with plasma from the seminal vesicles and with other fluids. The sperm “maturation” which includes “capacitation” that follows ejaculation is necessary for sperm to achieve fertilizing ability. A number of membrane changes are associated with these processes (Bearer and Friend (1990) J Elecon Micros Tech. 16: 281-297).

Sperm Cell Surface Markers

As described herein, the methods of determining when a desired trait is prevalent, and/or optionally differentially expressed by semen in an individual semen sample of interest is determined in part by an assay in which the expression of specified marker(s) is monitored over time. Thus, the metabolic status of semen is reflected by measurement of a marker. In one embodiment, the marker is component of the cell surface of the sperm, e.g., phosphatidylserine. The percent of individual sperm in the semen collection having a high phosphatidylserine concentration can be used as a cell surface marker of a metabolic status of sperm which occurs at a predetermined amount of time before the semen have the desired trait(s). In other embodiments the cell surface expression of marker is assessed using a lectin, an oligosaccharide conjugated to a fluorphore, antibodies, a positively charged protein conjugated to a fluorophore, merocyanine 540, YOPRO-1, or combinations thereof.

In another aspect, membrane permeability reflects changes to the cell surface. Thus, an indicator of the degree of membrane permeability can be reflected in the degree to which molecules cross the membrane and enter the sperm, e.g. the acrosome or the head, and/or become absorbed to the sperm cell surface. These molecules include, but are preferably not limited to, small molecules, dyes, and enzymes. These molecules, in particular the enzymes, can optionally be used together with either soluble or insoluble chromogenic or fluorogenic substrates to create a detectable signal. In another aspect, the cell surface changes can be visualized, either directly or through the use of morphological markers, through the use of brightfield (“light”) microscopy, and other forms of microscopy, including, but not limited to, phase contrast microscopy.

When antibodies to sperm cell surface markers are used to detect cell surface changes, they can be directly conjugated to a fluorophore or an enzyme. For example, a primary antibody conjugated to an enzyme in conjunction with the enzymatic substrate to produce a colorimetric reaction can be used in the methods described herein.

In some embodiments, the enzyme itself in conjunction with the enzymatic substrate can be used in the absence of an antibody.

Alternatively, a second antibody reactive to the first antibody can be used to increase the sensitivity of detection. The second antibody also can be directly conjugated with a fluorophore or an enzyme. Thus, the percentage of sperm positive (% positive), for binding of the antibodies the cell surface molecule, is in some instances assessed using a primary antibody in conjunction with a secondary antibody that is conjugated to a fluorophore or other detectable label. Numerous suitable antibodies are described in the literature. For example, a monoclonal antibody to human germ cells has been described by Naz et al. (1984; Science 225: 342-344), Saxena and Toshimori report a monoclonal antibody to MC31, a cell surface protein that is modified and redistributed during capacitation (2004; Biol Reprod 70:993-1000), Mor et al. describe membrane protein that binds heparin (HBSM) (2007; Biochem Biophys Res Com 352: 404-409) and Focarelli et al. report that a CD52 antibody presents a different result compared to an anti-gp20 antibody (1999; European Society of Human Reproduction and Embryology 5:46-51). A monoclonal antibody, 4B12, has also been reported that recognizes a surface membrane-associated protein located in the acrosomal portion of the spermatozoa that becomes accessible after capacitation (Mollova et al. (2002) Folia Biologica (Praha) 48: 232-236). Other molecules for use as markers during the capacitation process for which antibodies can be made are presented in Cohen-Dayag and Eisenbach (1994; Am J Physiol Cell Physiol 267:C1167-C1176).

In addition, antibodies that are not unique to sperm cells can be used to monitor sperm cell surface changes. For example, antibodies directed to the antibiotic cloxacillin and antibodies directed to the Calcium binding protein human calponin can be used, as well as antibodies to Salmonella species (Difco Salmonella H antiserum A-Z product number 224061). In some instances, if a rabbit anti-salmonella antibody is used as a first antibody, a goat anti-rabbit IgG can be used as a second antibody. In cases where an enzyme reaction is used to visualize binding, the second antibody is conjugated to an enzyme and its substrate is added as a solution. Nonlimiting examples of enzyme-substrate pairs are peroxidase/hydrogen peroxide, glycosidase/4-methyl-umbelliferyl-glycoside and horseradish peroxidase/TMB (3,3′,5,5′-tetramethylbenzidine).

In one embodiment, the assay involves obtaining a semen sample, incubating the semen sample, taking at least one sample or aliquot from the semen sample, assessing sperm quality by contacting the sperm sample with at least a first molecule or ligand that interacts with a cell surface molecule or component, determining the percentage of sperm positive (% positive) for binding the cell surface molecule, optionally determining the point at which the % positive begins to decline, terminating the incubation of the semen sample after a predetermined time measured from the jump point, and processing the semen sample for immediate use or for storage. Semen may be stored in straws, or otherwise, for artificial insemination. The cell surface molecule being detected may be present on the cell surface, and/or may have been previously cryptic, but now accessible for detection through permeability changes or lipid flip-flop (translocation) across the membrane.

With respect to assessing the ability to produce a sex bias in generated offspring, since the timing of many of the changes that occur during maturation or capacitation may occur at a different rate in X-bearing sperm versus Y-bearing sperm, these sperm cell changes, (including changes on the cell surface and/or internally) can be monitored to assess the ability to produce the highest sex bias in generated offspring. This is done by identifying the time at which the largest group of X-bearing sperm will be at their peak during the critical time post-artificial insemination with respect to fertilizing performance compared to the Y-bearing sperm. Thus sperm cell surface changes allow one to assess the point at which the highest sex bias can be generated upon insemination of, e.g., cows as well as heifers.

Exemplary Detailed Protocol

An outline of two exemplary detailed protocols for obtaining a semen sample having a gender bias using antibodies to salmonella are described as follows.

The first protocol uses light microscopy and fluorescence microscopy.

1. Treat aliquot of incubating semen sample

-   -   i. Into 1.5 ml tube, pipet the following and mix as directed:     -   ii. 100 ul GREEN 1     -   iii. 20 ul RED 2     -   iv. 5 ul BLUE 3, where one or more of the reagents contains a         molecule which binds to, or is incorporated in, sperm or a         fragment thereof, such as annexin V. mix     -   v. 5 ul neat semen, mix gently

2. Incubate treated aliquot

-   -   a. Place tube in dark for 20-30 minutes

3. Wash

-   -   a. Add 1 ml BUFFER     -   b. Microfuge 20 seconds     -   c. Carefully remove supernatant with 1 ml pipet

4. Score

-   -   a. Add ˜200 ul BUFFER to cell pellet and mix gently to resuspend     -   b. FOR MICROSCOPE: Transfer ˜5 ul to slide and score # positive         sperm (green fluorescence on head) and #total cells. Count at         least 100 cells. Calculate % positive. (% Positive=[# positive/#         total cells]×100)     -   c. FOR CYTOMETER: place aliquot of resuspended cells into         cytometer tube and analyze on a calibrated cytometer using the         methods described herein.

5. Determining time point for processing semen sample

-   -   a. Plot percentage of positive cells. When percent positive         increases sharply (usually doubles from one time point to the         next) in the timeframe of 3-6 h post-collection, that is the         assay jump point (time zero=time of collection).     -   b. Wait 2 h after the jump point, and process semen in the         standard protocol, with the following change: Ensure that         extender is cooled to 12° C. before it is added.         FIG. 13 provides data obtained with cattle using the above         protocol showing that metabolic status changes with age.         The second protocol continues with assessing the staining of the         treated aliquot using a cytometer         Before running this assay, be sure that semen has been collected         and incubated in a manner to minimize process failures.     -   6. START EQUIPMENT         -   i. Turn on computer         -   ii. Turn on cytometer         -   iii. If needed, empty waste bottle and fill sheath bottle     -   7. OPEN TEMPLATE AND NAME FILE         -   a. Click appropriate Template         -   b. Select File>Save CFlow file as . . .         -   c. Name file by date by typing date as yyyymmdd under File             Name: (e.g., for Jul. 18, 2009 type: 20090718)         -   d. Click Save     -   8. COLLECT DATA         -   a. Under the red Collect tab, click on desired cytometer             grid (A1 is for first sample, first bull, A2 is for second             sample, first bull. B1 is for first sample, second bull,             etc.)         -   b. Next to cytometer grid (e.g., A01) type sample             information (e.g., RDU-0 for first time point from bull RDU)         -   c. Check that stoplight shows green color. Load sample onto             SIP tube on cytometer and pull out plastic tube support             underneath tube         -   d. Click Run         -   e. After data are acquired, adjust vertical gate on             histogram plot (Plot 3) so that the percentage of positive             sperm (the peak on the right) can be determined. Record this             number. Remove sample from SIP tube         -   f. For the next sample, repeat process above starting with             step a         -   g. After the samples for that hour are finished, click             Backflush, wait for stoplight to show green, then click             Unclog     -   9. CLEAN AND SHUT DOWN EQUIPMENT         -   a. As desired throughout day, place towel under SIP tube and             select Backflush or Unclog         -   b. At the end of the day, place a tube of water on the             cytometer, select well H11 and click Run. Allow water to             clean system for at least 2 minutes. From the pull-down             menu, select Instrument>Run cleaning fluid cycle         -   c. Turn off cytometer, then turn off computer

Visualization of the sperm cell changes, whether at the surface or inside the cell, can be accomplished in numerous different ways. As discussed above, visualization based on the use of a fluorophore or other light emitting molecule, used alone or in combination with a binding protein or antibody. Suitable fluorophores include, but are preferably not limited to, carboxifluorescein acetate, phycoerythrin, calcein acetate, alexa fluor 488, YO-PRO-1, SNARF-1, and combinations thereof. In some embodiments combinations of the fluorophores are used where the range of excitation is similar (e.g. 488 nm), but emission occurs in different areas of the spectrum (e.g. the green wavelength (515 nm) vs. the red wavelength (610 nm)). The use of multiple fluorophores is particularly useful when more then one marker is being monitored.

The metabolic status of cells can be determined as a function of the cell's permeability to any molecule, such as a dye or stain or other molecules such as enzymes having insoluble chromogenic substrates. Suitable dyes for use in an assay to determine permeability of the cell membrane, or the ability of the cell to pump out or concentrate molecules including but are preferably not limited to, dyes, Annexin-V, Annexin-V-Biotin, biotin, Annexin V-PE, Annexin V-FITC, SAv-FITC, 7-AAD, Hydroethidine, Evans blue, chlorazol Black E, Coomassie Blue and Trypan blue, to name but a few. Combinations of these dyes can also be used. Chromogenic substrates include TMB (3.3′,5,5′-tetramethylbenzidine) for peroxidase, ABTS (2.2-Azino-di(3-EthylBenzthiazoline Sulfonic acid) for peroxidase, pNPP (p-nitrophenyl phosphate, disodium salt) for alkaline phosphatase, and 5-bromo-4-chloro-3-indoyl-beta-D-glucuronide (BCIG) for beta galactosidase. Also useful are carboxyfluorescein and phycoerythrin, calcium acetate, Yo-PRO-1, SNARF-1, AlexaFluor-488 and Fluorescein isothiocyanate (FITC).

Below are detailed protocols for analyzing metabolic changes reflected by changes in membrane permeability of the sperm and/or fragments thereof. The readout is in terms of the quantity of small bright particles similar in size to the particles comprising the punctate staining pattern—the fluorescent crescent—over the acrosomal region of intact sperm.

TREAT

-   -   Into 1.5 ml tube, pipet the following and mix as directed:     -   100 ul GREEN 1     -   20 ul RED 2     -   5 ul BLUE 3, mix     -   5 ul neat semen, mix gently

INCUBATE

-   -   Place tube in dark for 20-30 minutes

WASH

-   -   Add 1 ml BUFFER     -   Microfuge 20 seconds     -   Carefully remove supernatant with 1 ml pipet

SCORE

-   -   Add ˜200 ul BUFFER to cell pellet and close tube     -   Mix tube vigorously. This can be done by holding tube top firmly         in one hand and striking tube at tube bottom with finger of         other hand. Strike tube at least 5 times. Alternatively, a         vortex mixer can be used.     -   Transfer ˜5 ul to slide and evaluate at least 3 fields for         appearance of small fluorescent particulates (as evidenced for         example by staining) and for torn or partially missing         fluorescent crescents on the heads of positive sperm.     -   Score assay: no punctate staining=1, slight=2, pronounced=3.     -   Determine time point for processing semen sample     -   Upon appearance of a small particle diffuse objects staining at         a score of 2 or higher, immediately carry out first step of         processing or extension by adding extender to the semen sample

Modification of Above Protocol

TREAT

-   -   Into 1.5 ml tube, pipet the following and mix as directed:     -   100 ul GREEN 1     -   20 ul RED 2     -   5 ul BLUE 3, mix     -   5 ul neat semen, mix gently

INCUBATE

-   -   Place tube in dark for 20-30 minutes

WASH

-   -   Add 1 ml BUFFER     -   Microfuge 20 seconds     -   Carefully remove supernatant with 1 ml pipet

SCORE

-   -   Add ˜200 ul BUFFER to cell pellet and close tube     -   Mix tube vigorously. This can be done by holding tube top firmly         in one hand and striking tube at tube bottom with finger of         other hand. Strike tube at least 5 times. Alternatively, a         vortex mixer can be used.     -   Transfer ˜5 ul to slide and evaluate at least 3 fields for         appearance of small fluorescent particulates (punctate staining)         and for torn or partially missing fluorescent green crescents on         the heads of positive sperm.     -   Score assay: no punctate staining=1, slight=2, pronounced=3.

DETERMINE PROCESSING TIME

-   -   Determine the hour when the staining score increases above 1, by         using a quantitative scoring system, such as described by Maxim         Mokin and Joyce Keifer, supra. Three hours after that time         point, end the incubation by carrying out the first extension.         (For example, if the staining score is 1 at hour 2 and increases         to 2 at hour 3, process semen sample by for example, adding         extender at hour 6—where hour zero is the time of collection.)         Use extender that is at 12° C. and keep collection cool while         processing.

The above protocols, and modifications thereof, have been used extensively by the present inventor with cattle. However, the assay can be used for other species, such as human, in a similar manner. In addition to work with cattle, analysis of human sperm revealed an identical stain morphology in a variant of the present assay. Samples of human sperm were provided from a healthy donor. An expert in sperm quality assessment examined a sample by light microscopy and determined that it was typical in morphology and motility. Next, an assay was performed on aliquots of neat semen, which were evaluated by bright field and fluorescence microscopy. To prepare sample, 100 ul of neat semen and 400 ul of phosphate buffered saline pH 7.0 were mixed and centrifuged for 2 minutes at 3,000 rpm in an Eppendorf 5415C minifuge with fixed angle rotor. Supernatant was aspirated and one additional wash of 400 ul Phosphate Buffered Saline (PBS) was performed. The cells were resuspended in 300 ul PBS. 100 ul of washed cells were transferred to a clean tube to which was added 150 ul PBS, 6 ul of 1 mg/ml murine anti-human calponin monoclonal antibody (gift from Dr. E. Mabuchi, Boston Biomedical Research Institute) and 12 ul goat anti-mouse IgM-AlexaFluor 488 (Invitrogen Corporation, Cat. no. A21042, 2 ug/ml). The tube was incubated at room temperature for 30 minutes.

Then 1 ml PBS was added and the tube was centrifuges in the minifuge. Supernatant was aspirated, 250 ul PBS was added and aliquots were examined by bright field and fluorescence microscopy.

A punctate pattern of labeling over the acrosomal region was noted, with some post-acrosomal labeling at the midpiece. In some cases, a small amount of very bright punctate labeling with weak midpiece labeling was noted. This pattern is similar to that noted with bull sperm in the present assay, with the exception that in the present assay labeling is brighter and is confined to the acrosomal region.

Other Markers of Cell Surface Changes

Sperm cell surface changes that have been reported and can be monitored according to the present assay including, but preferably not limited to increases in net negative surface charge (Bedford (1963) Nature 200: 1178-1180; Yanagimachi et al. (1972) Am J Ant. 135:497-520; Lopez et al. (1989) Gamete Res. 18:319-332), changes in glycoprotein amount or localization (Baker et al. (2004) J Andrology 25:744-751), changes in cholesterol and lipid distribution (Wolfe et al (1998) Biol Reprod 59:1506-1514; Flesch et al (2001) J Cell Sci 114:3543-3555) and phosphatidylserine location (Pena (2007) Asian J Androl 9:731-737). For example, since sperm develop a net negative surface charge over time, positively charged proteins can be conjugated to an appropriate fluorophore for evaluation. Suitable proteins have a pI greater than or equal to 8.5 so that they will be positively charged at the pH of the binding assay and include histidine-rich proteins such as the late embryogenesis abundant (LEA) proteins (Moons et al. (1995) Plant Physiol 107:177-186). Similarly, lectins conjugated to a fluorophore can be used such as Pisum sativum lectin (PSA), tomato lectin (LEA), peanut lectin (PNA), Aleuria aurantia agglutinin lectin (AAA), Ulex europaeus agglutinin lectin (UEA-1), wheat germ lectin (WGA), Solanum tuberosum (STA) and Tetragonobolus lectin (TPA). Mono- or oligosaccharides suitable for conjugation to a fluorophore include those terminating at the non-reducing end in fucose, galactose, or mannose, or being polymers of lactosaminoglycans.

Other Markers

Alternatively, the metabolic status of semen is assayed by markers assessing sperm quality. One marker of sperm quality is measured on the basis of sperm motility grade and percent. Another marker of sperm quality is measured on the basis of the number of intact acrosomes.

Incubation Conditions

The methods of monitoring the metabolic status and/or desired traits of the semen sample involve are performed as the semen sample is incubating. Typically, the semen sample is incubated in a suitable collection container which has an overall and uniform temperature equivalent to the ambient temperature or falling within some range with a low temperature not lower than 0° C. and a high temperature not higher than the body temperature of the pertinent species or the ambient temperature. In one embodiment the semen sample is cooled from a collection temperature of approximately 35° C. to 12° C., where the incubation is then maintained at a constant temperature of 12° C.+/−0.1° C. In other embodiments the sample may be cooled from a collection temperature of approximately 35° C. to any of the following temperatures (+/−0.1° C.) at which the incubation is maintained at a constant temperature until the semen sample is collected for use, e.g., packaging into straws: (34° C., 33° C., 32° C., 31° C., 30° C., 29° C., 28° C., 27° C., 26° C., 25° C., 24° C., 23° C., 22° C., 21° C., 20° C., 19° C., 18° C., 17° C., 16° C., 15° C., 14° C., 13° C., 12° C., 11° C., 10° C., 9° C., 8° C., 7° C., 6° C., 5° C., 4° C., 3° C., 2° C., 1° C., 0° C., and any temperature point in between. In alternate embodiments, the variation of the constant incubation temperature includes, but preferably is not limited to, +/−0.1° C., +/−0.2° C., +/−0.3° C., +/−0.4° C., +/−0.5° C., and +/−1.0° C. The devices disclosed in application PCT/US09/38134 or in US 2006/0147894 A 1 are examples of suitable collection containers, as is the device disclosed in U.S. Pat. No. 5,895,749. Application PCT US09/38134 is hereby incorporated by reference in its entirety. In one embodiment, the incubation temperature is not held constant, but varies according to a defined pattern or cycle of temperature intervals, the incubation at each interval being maintained at a specified temperature for a specified duration.

In one embodiment, the methods described herein provide a means for determining when to process semen samples, for example, by extending and aliquoting the semen sample into straws for freezing or immediate use, e.g., in artificial insemination. Various reagents and physical conditions to which sperm may be exposed during processing, can affect sperm metabolism. The present assay also provides a means of evaluating treatments most beneficial to sperm and enabling users to improve sperm quality and properties in commercial operations by use of an optional treatment. The treatment minimizes variability in the sperm microenvironment within the semen ejaculate. For example, variability can result from the fact that, in some species, different pulses of ejaculate have very different biological compositions and, consequently, there is a gradation within the chemical microenvironment. In other cases the variability is a result of temperature or chemical differences within the semen sample. Therefore, the treatment that homogenizes the semen collection can comprise contact with chemical agents such as, for example, glycosidases (e.g. sialidase, galactosidase, glucosidase, and/or mannosidase), perioxidase, and/or horseradish peroxidase), proteases (e.g. chymotrypsin, trypsin, and/or elastase), lipases (e.g. phospholipase C and/or phospholipase A) and kinases (e.g. diglyceride kinase, glycogen synthase kinase and/or inositiol kinase), to name but a few, and/or mono- or oligosaccharides, etc., or incubation at particular temperatures. Suitable temperatures can be any temperature between 0° C. and the ambient and/or the body temperature, or for example, between 0° C.-40° C., 15° C.-40° C., 18° C.-25° C., 25° C.-30° C. 4° C.-40° C., 15° C.-40° C., 18° C.-25° C., or 25° C.-30° C., to name but a few. However, preferably, incubation is maintained at a preselected temperature ±1° C., or ±2° C. Treatment can also comprise gentle mechanical mixing such as slow rocking of the tube, use of a rotary shaker, or tube inversion Combinations of any of the above treatments that homogenize the semen environment are also envisioned.

Mono- or oligosaccharides can be added to the semen collection sample after having been equilibrated to the same temperature as the collection sample. Ideally, this is done at the time of collection, but can be done at any time during the incubation step. Suitable mono- or oligosaccharides are carbohydrates that resemble those present in the isthmus. It is known that the isthmus of the oviduct can serve as a site of sperm binding to create a reservoir of sperm, which are presumed to be in a stabilized state. The mono- or oligosaccharides are chosen to effect membrane stabilization, which stabilize sperm against the manipulations they must undergo. Suitable monosaccharides include sialic acid, mannose, fucose or galactose, while appropriate oligosaccharide polymers are linear or branched chains of any compatible sugar or glycoproteins carrying carbohydrate chains that terminate at their non-reducing end in fucose, galactose, sialic acid or a combination of these sugars. Additional suitable treatments include commercial semen diluents and extenders, TALP, glycerol, egg yolk, bicarbonate ions and calcium ions or calcium chelators. Combinations of the above can also be used.

Additional treatments include the addition of a sugar polymer, a lipid, an enzyme or combinations thereof is added. Another treatment changes the temperature of the semen sample. Yet another treatment is mechanical agitation of the semen sample. In some embodiments multiple treatments of the same or different types are performed at the same time or are preformed sequentially.

With respect to the treatment, one embodiment envisions a treatment where a sugar polymer, a lipid, an enzyme or combinations thereof is added. Another embodiment uses a treatment that changes the temperature of the semen collection. In yet another embodiment the treatment is mechanical agitation. In some embodiments multiple treatments of the same or different types are performed at the same time or are preformed sequentially.

Fertility

The field of male factor infertility has reached a threshold where advance requires a novel approach. Sperm dysfunction is reported as the most common cause of infertility (Barrett et al., 2011). Existing assays do not provide the predictive ability that is needed as the following excerpts indicate:

-   -   “The diagnostic tests currently used for semen are inadequate .         . . . ”, (Miller, 2011);     -   “Fertility is one of the most economically important traits         controlling animal reproduction. Despite its significant         economic impact, there are no reliable markers to predict semen         quality”, (Wang et al., 2011);     -   “Traditionally, the diagnosis of male infertility has relied         upon microscopic assessment and biochemical assays to determine         human semen quality . . . . However, none of these parameters         addresses sperm function and their clinical value in predicting         fertility is questionable”, (Lewis, 2007);     -   “Antisperm antibodies (ASA) may be an important cause of         infertility but current tests for the detection of ASA have poor         prognostic value”, (Chiu and Chamley, 2002); and     -   “We review the current state of the field and provide insights         for further development. We conclude that: (i) there is little         to be gained from more studies identifying/characterizing         various populations of men using a basic semen assessment . . .         . ” (Barratt et al., 2011)         The pessimism of these experts in the field of male factor         infertility is understandable. Existing assays fail to measure         key parameters across time. Freshly ejaculated sperm can't         fertilize, they must acquire that capacity.

However, several aspects of Applicant's invention disclosed herein overcome these limitations of the art, including the use of (1) Applicant's novel dynamic real-time assays to measure the metabolic status of sperm and (2) Applicant's novel methods to stabilize this metabolic state at a desirable point to achieve optimum performance.

Male Factor Infertility and Assays

Sperm dysfunction was identified as the single most common cause of infertility by Barratt at al. (2011). Currently, male infertility evaluation and judgment is based on an assay based on a single point in time. The snapshot is considered representative of the sperm from that particular male. Yet Applicant has discovered as described in animal experiments herein that fertility varies with the metabolic status of the ejaculate, and varies across time in every ejaculate (FIG. 23), even among different ejaculates obtained from the same individual. To Applicant's knowledge, this variation has not previously been taken into account in evaluating male fertility, as described in the inventor's assays described herein.

Types of Assays and Model of Sperm Biology

All of the assays summarized in FIG. 1 have been used as research tools to evaluate the changes in sperm biology, termed capacitation, that occur with time. But these assays have not been applied repeatedly in real time with a fresh ejaculate, detecting sperm metabolic status in real time in order to optimize the ejaculate's performance in insemination. Applicant's disclosure herein is believed to be the first to teach the use of multiple real-time assays and the data obtained from them to adjust sperm processing to optimize sperm performance prior to insemination.

It has been recognized in the art that freshly ejaculated sperm are infertile; they must undergo maturation before they can fertilize an egg.

FIG. 17 illustrates the art recognized process of maturation of sperm. The process of maturation, referred to as capacitation, includes a number of biochemical processes, as reviewed by Flesch and Gadella, (2000). Ejaculated sperm first swim to and bind to oviductal sperm reservoirs, from which they are released over time (Revah, 2000). After the maturation step that releases them from the reservoir, punctate fenestrations form in the plasma membrane (D. Miller, personal communication), through which the underlying acrosomal membrane—postulated to possess egg receptors—appears. Hybrid vesicles of both membranes are extruded from the cell in a fashion believed to assist in binding and penetration of vestments surrounding the egg (Buffone et al., 2009). This extrusion also allows exposure of acrosomal contents and inner acrosomal membrane (Yanigimachi, 1981), containing structures and enzymes that facilitate egg binding and fertilization (Jones and Williams, 1990). In vitro, small vesicles are shed from the acrosome, containing hybrid mixtures of acrosomal and plasma membranes that appear in the semen (Primakoff et al., 1980). In vivo, ultimately the acrosomal contents are lost during binding to the complex layers of vestments that surround the egg (right). Sperm then interact with the egg zonapellucida and egg plasma membrane, penetrate and fertilize (Jin et al., 2011; Yanagimachi, 2011). See FIG. 18 for biochemical pathways of capacitation (Salicioni et al., 2007).

Coordination of sperm capacitation with egg binding and fertilization as described by Flesch and Gadella, 2000. FIG. 17B illustrates the art recognized sequence (steps (A)-(F) of mammalian fertilization. (A) Freshly ejaculated sperm cells are activated in the female genital tract during a process called capacitation. (B) Capacitated sperm cells are hypermotile and are able to bind to the egg extracellular matrix (ZP). (C) Binding of sperm cells to the ZP triggers the acrosome reaction and acrosomal enzymes are secreted. (D) Hydrolytic enzymes secreted from the acrosome degrade the ZP and subsequent sperm cells penetrate the ZP, enter the perivitelline space and bind to the oolemma with the apical tip. (E) Subsequent to apical tip binding, oolemma binding changes to the hairpin structure of the acrosome reacted sperm cell. (F) After hairpin structure binding to the oolemma, the sperm cell fuses with the oocyte and the sperm cell is subsequently incorporated in the oocyte. 1: perivitelline space; 2: ZP; 3: oolemma (egg plasma membrane). Additional art recognized detail of fertilization is provided in FIG. 17C, as described by Ikawa et al., 2010.

Capacitation is art recognized to comprise both fast events and slow events, as illustrated by FIG. 18A and FIG. 18B, Salicioni et al., 2007. Salicioni and her colleagues only disclose two assay targets and only on the fast events area. They do not disclose the use of these assays in real-time to control the timing of sperm processing.

In the clinic, assays are run at a single time point, and so again are not reflective of optimal biology, except by accident of measurement timing. In contrast, Applicant's assays disclosed herein are real time assays encompassing multiple time points. The instant inventor's assays disclosed herein encompass numerous biomarker targets, including, but not limited to membrane changes that affect diffusion and uptake of molecules such as dyes, antibodies, lipids, and lipid based molecules for example, into sperm, motility changes, and expression of previously-cryptic membrane components during membrane rearrangements. Some membrane changes result in changes in permeability to molecules of different size and/or composition, as well as changes in membrane receptor expression, composition and/or conformation. These changes are reflected by Applicant's use of antibodies, lectins, and dyes, Annexin V and other molecules in real time assays of sperm samples. See for example, FIG. 23 and Table 1. Other biomarkers targeted in the assays described herein include, but are not limited to the following: fluidity changes that occur with capacitation, measured for example, through the use of membrane fluidity probes, pH changes that occur with capacitation, measured for example, through the use of pH probes, and changes in acrosomal ruffling that occur with capacitation, measured for example, through optical measurements of acrosomal ruffling. See for example, FIGS. 21 and 22. Additional biomarkers targeted in the assays described herein include, but are not limited to the following: the cell number, cell morphology, motility, ejaculate volume and sperm agglutination as measured, for example, by antibodies.

Model of Sperm Biology & Maturation into Ability to Fertilize

Historical studies have led to a model of sperm biology having the following characteristics: As discussed above, it is art recognized that freshly ejaculated sperm acquire ability to fertilize an egg through a process known as capacitation (Austin, 1951; Chang, 1951). These historical studies include assaying capacitation by monitoring the ability of sperm to undergo the acrosome reaction. The data from these historical studies indicates that acquisition of this capacity occurs at a time unique to each ejaculate and that once sperm are activated, they have a very short life, on the order of hours (Cohen-Dayag et al., 1995).

Because the availability of sperm is required over the entire period when the egg may appear, a steady stream of active sperm must be maintained through activation of sequential maturing cohorts. This means that fertility of sperm used in assisted reproductive technologies may fluctuate both as the first cohort of sperm activates, and as sequential cohorts arise, mature, and die.

It is art recognized that a mammalian ejaculate acquires the fertilizing ability at a range of times from 0.5-3.3 hours post-ejaculation, with exact timing unique to each ejaculate. The data in FIG. 19 which illustrate this variability in acquisition of fertilizing ability was generated using sperm which were collected and from which samples were evaluated at various times post-ejaculation. One size does not fit all, as shown by differential curves that highlight a fast- and a slow-maturing ejaculate.

Specifically, FIG. 19 shows human donor sperm kinetic against the EnGender assay and other biomarkers run simultaneously on the same ejaculate, with ejaculates from different donors. Human semen was collected from different donors into a specimen cup in a pre-warmed insulator at 32 C (a plastic cup with a weighted metal base, the idesign cup designated as “clear plastic/chrome tumbler” (Target, online item number 695107, Store Item Number (DPCI): 064-05-2111), which could receive the specimen cup and then be transferred to a 12 C water bath and float the insulator and collection cup). Within less than 5 minutes after ejaculation, the semen sample in cup and insulator was placed in a 12 C chiller bath and sampling/assaying was begin immediately and continued for hours at either 15 min or 30 min intervals, as described in the SOP for 30 minute intervals. In the designated cases, the sampling frequency was increased. Top panel, examples of profiles from 3 different donors (1, 2 and 5) generated by the antibody assay, run as described in the assay standard operating procedure (SOP) included in this specification, except that samples were destroyed after use, not preserved for insemination. Middle and lower panels show three different biomarker assays—involving addition of either antibody, Fc fragment, or heparin-FITC—run on two different donors. The antibody assay is shown as a benchmark and run as described (for all reagents and method except the Fc fragment and heparin-FITC as described in this figure legend, see assay SOP). For the Fc assay, the process was run as described in the SOP appended to the specification, with the following change: the Red 2 solution of primary antibody was omitted and for it was substituted 20 ul of Chromepure Rabbit IgG Fc Fragment 2.2 mg/ml (Jackson ImmunoResearch Laboratories, Inc., code 011-000-008). For the heparin assay, the process was run as described in the SOP appended to the specification, with the following change: the Red 2 solution of primary antibody and the Blue 3 solution of secondary antibody were omitted and for them was substituted 5 ul of heparin-FITC (Heparin, Fluorescent, from Polysciences Inc., Cat #16092, supplied as 10 mg dry powder and resuspended before use to a volume of 1 ml in a 1× solution of PBS buffer disclosed in the SOP. The 1× buffer solution had been made to the buffer tablet manufacturer's specifications as listed on the buffer tablet bottle).

Applicant's real time assays for optimizing sperm performance before processing for insemination accommodate sperm biology and are based on the observation that late-maturing collections (right red arrow) could be rejected for quality if assayed immediately—when in fact they simply gain fertility slowly.

A possible example of how a slow gain in fertility can influence fertility perception is shown in the Table 9 below (Kobyra and Cohen, unpublished results). Note that the percentage of sperm that is motile at maximum motility is the same in straws from both the passing and failing ejaculates. The only difference is when in time the maximum motility is detected.

TABLE 9 Slow gain of motility by sperm can be perceived as poor semen quality. Frozen straws of bull semen were obtained from ejaculates that passed or failed quality control. Straws were thawed and sperm were evaluated by swim-up at various times post-thaw, to determine the percentage of motile sperm in the population (measured as the percentage of sperm capable of swimming up). Sperm were quantitated by hemocytometer counting by brightfield microscopy. % Motility, time post-thaw of testing Ejaculate QC result 0 h 1 h 2 h Passing 41 24 24 Failing 12 12 42

The instant invention differs from previous assays described in the art in that the assays of the inventor described herein are rapid assays, and are run at multiple time points on fresh ejaculates with T₀ beginning immediately after collection, to enable use of sperm in their optimal metabolic state relative to a desired performance.

The timing of the optimal metabolic state with respect to the timing for processing the semen sample may differ according to the property/performance being evaluated, e.g., fertility, gender bias, etc. Fertility improvements were produced when the assay was run in the mode that optimizes fertility (FIG. 23). More female calf births on dairy farms were produced when the assay is run in the mode that creates gender bias (FIG. 23). These modes differ in the timing of sperm processing relative to biomarker status (see Working Examples 16-20 for nonlimiting Standard Operating Procedure for the Assays described herein). Once biomarker data are obtained, the timing of sperm processing is adjusted such that sperm are stabilized in a state that enhances fertility, or in a state that creates gender bias. The post assay stabilization is achieved by steps in the processing of sperm prior to insemination.

The instant invention also offers potential to evaluate the status of frozen sperm, to predict their performance attributes prior to insemination. In such an application, the invention is run as a single assay on thawed straws of frozen semen, and calibrated against one or more curves measuring biomarker expression over time in sperm capacitation.

Antibody-Based Reagents for Use in Detecting Biomarkers in Real Time Rapid Assays

Application of the instant invention in a preferred embodiment using antibodies to assay biomarker expression, in particular an Fc receptor biomarker, to optimize sperm fertility in humans or gender bias in cattle, is shown for human Assisted Reproductive Technology (ART) (FIG. 20A) and for frozen doses of cattle semen used on dairy farms (FIG. 20B). The preferred embodiment of antibody-based assays are run by mixing a 5-10 ul sample of semen with the antibody assay reagents, incubating about 15 minutes, washing, and scoring by microscope or cytometer (Gatza et al., 2011) with total elapsed time of less than 30 minutes sampling to score. In this way, the assay is rapid enough to be run multiple times at closely spaced intervals, allowing calibration with standard curves before insemination.

Use of Acrosome Based Biomarkers in a Real Time, Rapid Assays

Acquisition of fertilizing ability by sperm involves profound real-time changes in the acrosome that begin post-ejaculation and continue for hours, as captured in still shots of a video by Silvestroni (2008) (FIG. 21). Acrosomal changes begin at left. The white regions that develop on the acrosome (dark part of sperm in image 1)—the tops of the wrinkles (2,3,4) and the vesicular materials (4,5) that develop during sperm maturation—appeared biomarker positive when freshly-ejaculated bull sperm were assayed in real time over a period of hours by the instant invention (Wrenczycki and Cohen, unpublished results). For example, the initial appearance of the acrosome was dark on most sperm, with the appearance of cells with labeling on the rostral portion of the acrosome in a punctate pattern. As the ejaculate aged, labeling of the peaks of what looked like mountain ranges or wrinkles on the acrosome occurred, and the acrosome became generally labeled, with some punctate labeling remaining. Older ejaculates had a proportion of sperm with highly labeled material partially or completely detached from the head and no longer coincident with the acrosome, but in some cases stuck to the middle of the sperm head at a single point.

Use of Microscopy—in Detecting Biomarkers in Real Time Rapid Assays

FIG. 22 illustrates a comparison of biomarker sperm status by scanning electron and fluorescent microscopy. To evaluate the relationship between sperm morphology and antibody-based Fc receptor biomarker status, bull sperm were assayed for biomarker and evaluated by microscopy. The same sperm were imaged after fixation by both fluorescent and scanning electron microscopy. The antibody biomarker is initially cryptic and is acrosome-associated (Slivestroni and Cohen, unpublished data). As sperm age, biomarker positive small vesicles appear (FIG. 25F) in the semen (Cohen et al., unpublished data), consistent with prior observation of acrosomal maturation producing shedding of vesicles (Primakoff et al., 1908).

Use of Antibody Based Real Time Assays Before Insemination to Increase Fertility in Cattle

Real-time assays were used before inseminations to optimize bull sperm performance as illustrated in FIG. 23. Bull semen was collected and processed as described in the attached Biomarker SOP. Human semen was processed as described in the attached OptiFert SOP. Assays were run at the frequency indicated on the X-axis. Human semen was collected from different donors into a specimen cup in a pre-warmed insulator at 32 C (a plastic cup with a weighted metal base, the idesign cup designated as “clear plastic/chrome tumbler” (Target, online item number 695107, Store Item Number (DPCI): 064-05-2111), which could receive the specimen cup and then be transferred to a 12 C water bath and float the insulator and collection cup). Within less than 5 minutes after ejaculation, the semen sample in cup and insulator was placed in a 12 C chiller bath and sampling/assaying was begin immediately and continued for hours. FIG. 23A, Top panel, examples of profiles from 4 different bulls, showing arrows corresponding to the different times post-collection at which each ejaculate reached a maximum percentage of biomarker cells. FIG. 23A, Bottom Panel, comparison of biomarker kinetics of antibody and dye assays on an ejaculate from one human donor. Assays were run as described in the OptiFert SOP, with the following change for the dye assay: Primary antibody, designated as Red 2, and secondary antibody, designated as Blue 3 (see SOP for detailed disclosure of antibody types and catalog numbers), were omitted. Both increased female calvings as illustrated in FIG. 23B at statistical significance (p<0.0001).

Further, cow fertility improvement were produced by real time assays with standard methods of cattle insemination as illustrated in FIG. 23C and FIG. 23D. FIG. 23C shows the production of statistically significant (p=0.10) increased fertility by the biomarker-half of 5 split ejaculates compared to control, on dairy farms. FIG. 23D shows the production of statistically significant (p=0.03) increased fertility of biomarker ejaculates compared to conventional ejaculates from the same bull, on dairy farms. Note: fertility improvement of 1-2% is valuable in agriculture—the instant invention shows improvement of 6.4-6.7%, The rate of female calf generation equals or exceeds existing technology, and provides the additional advantages of no semen discard from elite bulls, ability to inseminate cows, not just virgin heifers, and ability to work with the small ejaculates produced by very young bulls, who are now brought into production so young because they are genetically proven to be good sires, not proven by births of daughters.

To evaluate attributes of semen based on different processing times post-biomarker maximum, collections were processed at different times relative to the biomarker maximum. Based on real time biomarker assay (using antibodies comprising the antibody constant region), extension and freezing times were adjusted to optimize sperm performance (see FIG. 20). Doses of frozen semen were prepared at standard cell numbers of 15-18 million cells/straw. These straws were used in standard artificial insemination (AI) of cows and heifers in working dairy herds. Fertility was determined by non-return rate and calf sex was determined at birth, from data in the Irish Cattle Breeding Federation database that was analyzed by the National Cattle Breeding Centre in Enfield, Ireland. Control sex ratio of calvings was drawn from Berry et al., 2011.

For human sperm, a donation was obtained, cooled and assayed in real time. For the human donation, instant invention biomarker assay methods using both antibodies and dye permeation were run.

Use of Dye in Real Time Assays Before Insemination to Optimize Performance

In one embodiment of a specific dye-based assay described herein is based on use of antibody diluent catalog number 10008, sold by Life Technologies. It contains bovine serum albumin, azide and a dye/dyes. The dye component is food coloring (Life Technologies technical service representative to Cohen, personal communication).

Optimization of Sperm Performance

FIG. 24 illustrates the timing of multi-point assays to optimize sperm performance. The assay curve (see, for example, the human sperm example of FIG. 23) is shown with the typical biomarker kinetics made visible by the assay, and with the outcomes that can be produced by selecting sperm at the biomarker status indicated (FIG. 24A). Cattle gender bias is produced by adding diluent and freezing cells about 2 hours after maximum biomarker expression. Fertility is produced by adding diluent and freezing cells about 1 h after maximum biomarker expression. FIG. 24B provides the actual assay from the instant invention showing maturation of multiple cohorts of sperm. Maturation of multiple cohorts of sperm as measured by the instant invention in two bulls, illustrating the difference in timing of maturation between the ejaculates. Both first and second cohorts were successfully used to create gender bias in cattle (FIG. 23).

Additional Biomarkers and their Sequential Order of Appearance During Sperm Maturation

FIG. 25 illustrates the use of additional biomarkers and their sequential order of appearance during sperm maturation. When working with sperm predictively, we found that sperm acquire optimal fertility before they acquire the ability to produce a female gender bias. This is consistent with our working model of predictive sperm biology, (FIG. 25). We therefore evaluated a number of biomarkers paired with our antibody-based assay targeting the Fc receptor biomarker, in order to determine the sequential order of biomarker appearance as sperm undergo maturation. These biomarkers include (A) CD46, (B) soybean trypsin inhibitor, (C) motility, (D) % acrosome reacted sperm, (E) dye permeation, and (F) appearance of biomarker positive vesicles in assay (by microscopy). Bull sperm were used for all but (E), which is based on human sperm. Instant invention biomarkers include a dye based-assay for clinical applications, and an antibody-based assay for veterinary applications. This data allows for calibration of data generated using one (or more) biomarkers from an individual semen sample in order to optimize performance of the individual semen sample.

FIG. 25. Biomarker kinetics, of different markers. Bull semen was collected and processed as described in the attached Biomarker SOP. The antibody assay is shown as a benchmark and run as described (for all reagents and method except the CD46, SBTI, motility, acrosome reacted sperm and vesicle assay, see assay SOP for veterinary application). Other markers were tested as detailed below. All assays are run at ambient temperature. In some cases, scoring by microscopy was substituted for cytometry. In these cases, at the end of the assay cells were diluted with the buffer of the SOP to an appropriate density, applied to slides, and examined under brightfield and also under mercury lamp illumination for fluorescence, using the FITC filters. Biomarker positive and negative cells were counted, and the percentage of biomarker positive cells was determined.

A. Antibody biomarker was run in comparison to an assay of CD46, a marker claimed to be present on sperm (note: discoveries of the instant invention support either binding of anti-CD46 antibody to CD46 antigen, or attachment of the anti-CD46 antibody via its Fc domain). For the CD46 assay, the process was run as described in the SOP appended to the specification, with the following changes: the Red 2 solution of primary antibody was omitted and for it was substituted mouse anti-CD 46 apntibody (BD Pharmingen purified mouse anti-human CD46 0.5 mg/ml), which was incubated for 20-30 min. 1 ml buffer was added and cells were washed as described per SOP. Washed cells were resuspended in Green 1 (antibody diluent) and 5 ul of Alexa-fluor 488-labeled anti-mouse IgG (Invitrogen) was added, with incubation for 20-30 min. Another wash was performed, and cells were resuspended in buffer for counting by microscopy. B. Assay of soybean trypsin inhibitor (SBTI) was conducted as per the vet SOP, with the following changes: Red 2 and Blue 3 were omitted, and 5 ul SBTI-AlexaFluor-488 conjugate (Molecular Probes) was added to Green 1. C. Assay of motility was conducted by a trained observed using microscopy. D Evaluation of acrosome-reacted sperm was performed by a trained observer using microscopy. E. Appearance of biomarker-positive vesicles was determined by a trained observer using fluorescence microscopy CD46 and soybean trypsin inhibitor

CD46 antibody recognition was evaluated by fluorescence microscopy in two ways that illustrate its distinction from other antibodies. In the first case, anti-CD 46 antibody was mixed with bull sperm, cells were washed to remove unbound antibody, and a fluorescently-tagged second antibody was added. In the second case, the primary and secondary antibodies were pre-mixed. The pre-mixed antibodies produced the strongest signal. This is in contrast to most other antibodies, where pre-mixing prevents formation of signal. Loss of signal is interpreted as resulting from competition between the sperm Fc receptors and the secondary antibody, where the secondary antibody would bind to Fc carbohydrates and block sperm binding. Most secondary antibodies are directed to Fc carbohydrates in order to enable a single secondary to recognize many primary antibodies possessing different FAb regions. The assays of the instant invention evaluate the process of capacitation itself, starting with uncapacitated, freshly ejaculated sperm assayed beginning within 5 minutes of ejaculation T₀. As sperm go through the unprovoked transition from uncapcitated to capacitated, the first biomarker of capacitation expressed on their surface is the ligand to CD46 antibody, then soybean trypsin inhibitor (SBTI). SBTI is a protease inhibitor that binds to acrosin, a protein that is released from the acrosome during exocytosis (Tollner et al., 2000). The sequence of CD46 expression followed by SBTI biomarker expression as sperm progress from an uncapacitated state into a capacitated state is in contrast to Harper's teaching that sperm could bind SBTI before becoming able to bind CD46 (Harper, 2008). Labeling for both SBTI and CD46 was in human sperm that were collected, allowed to liquefy for 30 minutes, then incubated in capacitating medium for at least 4 hours and then provoked to undergo acrosome reaction by addition of ionophore.

Motility and Acrosome-Reacted Sperm

Kinetically, it appears that freshly-ejaculated bull sperm gain motility as evaluated by microscopy before expressing the antibody-based Fc receptor biomarker (FIG. 25C). As an ejaculate ages an increasing number of acro some-reacted sperm accumulate (FIG. 25D). During the acrosome reaction the contents of the acrosome are released outwardly. The cell membrane of the spermatozoon fuses with the outer membrane of the acrosome. The contents of the acrosome flow out through the resulting pores.

Applicant's findings described herein indicate that, in contrast to what has been published, acrosomal integrity does not correlate linearly with fertility. At time zero (T₀) (the first point assayed immediately after ejaculation), 15% of bull sperm are acro some reacted and sperm have not gained full fertility (FIG. 23C). The fertility optimization associated with the antibody-based Fc receptor biomarker assay occurs about 1 hour after biomarker expression peaks (FIG. 23C), a time associated in FIG. 25D with 37% of sperm acrosomally reacted. This means fertility is maximal—not when sperm show intact acrosomes—but when the number of acrosome reacted sperm has increased 2.5-fold, a finding is novel, having not yet been described to the best of the instant inventor's knowledge. Measuring the percentage of sperm that have intact acrosomes is a standard test for fertility in veterinary and clinical practice, but the data disclosed herein indicate the correlation with fertility is poor. This may be one of the reasons so many experts agree that present semen diagnostics are inadequate as described in the background section.

Dye Binding, Antibody Binding, Vesicle Appearance and Other Labeling Patterns

Changes in dye binding have been widely documented, although they have not been applied in multipoint assays immediately post-ejaculation to improve sperm performance. The dye employed here, examined by AmnisImagStream cytometer, produced patterns more complex than antibody binding (Barteneva and Cohen, unpublished results). We observed that antibodies label initially the thin anterior acrosomal crescent, appearing identical to the Fertility Associated Antigen (FIG. 26A) of Sprott et al. (2000) and identical to the region to which solubilized egg zonapellucida proteins bind (FIG. 28). As the ejaculate ages, labeling extends to half-head labeling of the entire acrosomal region (FIG. 26). While vesicles have been noted to appear as acrosomal exocytosis precedes (Primakoff et al., 1908), we are the first to observe vesicle labeling with the instant invention biomarkers (FIG. 25F).

FIG. 26 provides photomicrographs of labeling with the following biomarkers: (A) Fertility-associated antigen (Sprott et al., 2000) and (B) Antibody biomarker, hours-old collection (Wrenzycki and Cohen, unpublished). Sperm labeled with the antibody marker immediately after ejaculation resemble the crescent labeling seen in (A). FIG. 10 also provides photomicrographs of labeling with the following biomarkers: (C) soybean trypsin inhibitor (Harper et al., 2008) and (D) CD46 (Harper et al., 2008). These micrographs show that only the antibody biomarker was evaluated with multipoint assays on a fresh bull ejaculate, with assay of sperm directly from an ejaculate that was cooled (no washing or other treatment).

Fertility-Associated Antigen (FAA)

Fertility-associated antigen (FAA) was identified as a heparin-binding antibody (McCauley et al., 2004). It was incorporated into a single-point cassette lateral flow assay (ReproTest) to discriminate highly fertile from subfertile bulls, but is no longer on the market. Labeling for both SBTI and CD46 was in human sperm that were collected, allowed to liquefy for 30 minutes, then incubated in capacitating medium for at least 4 hours and then provoked to undergo acrosome reaction by addition of ionophore.

The biomarker temporal map, illustrated in FIG. 27 shows how biomarkers for the multipoint assay are chosen across the capacitation time spectrum of sperm. Sperm images map one preferred embodiment, the antibody-based assay, onto the maturation of a cohort of sperm. Since active sperm have a limited life (Cohen-Dayag et al., 1995), sequential maturation of cohorts is required to produce a steady supply of activated sperm for when the egg appears. The arrangement of biomarkers across the temporal maturation of sperm is enabled by the real time, multi time point assays described herein, which uses the timing of other markers relative to expression of the instant invention antibody assay biomarker to normalize ejaculates to each other. We have already shown that the timing of maturation/capacitation of each ejaculate differs, which is why one cannot orient biomarkers across a timeline without a gold standard used to normalize ejaculates to each other. When this is done, it indicates the order of biomarker expression shown below, mapped against sperm heads showing the type of signal produced by the instant invention antibody-based assay, relative to timing of other biomarkers' expression profiles.

The endogenous ligand for the antibody-based Fc receptor biomarker is a Fc receptor on sperm. The instant inventor has shown that the Fc marker/biomarker is present as a relatively late biomarker in a punctate pattern associated with the acrosome. Though not being bound by theory, current thinking suggest that fenestrations in the plasma membrane that enable acrosomal content exposure occur after sperm dissociate from storage reservoirs in the oviduct and are making their way to the egg (Miller, D. J., personal communication). Binding is inhibited by fucoidan, and sulfated glycans are thought to be part of the egg vestements (Topfer-Petersen et al., 1988). Release of acrosomal vesicles (hybrid with plasma membrane components) is thought to be part of the mechanism by which sperm burrow into the egg vestiments. Sperm are thought to attach to the egg vestements, releasing vesicles bound to egg vestements as they burrow deeper, so that they are not anchored to the initial superficial site of attachments (Buffone, 2009). Applicant's data shows the Fc marker/biomarker is typically found initially in a punctate pattern on sperm, and later in small vesicles found only in aged ejaculates.

Proteins from the zonapellucida of the egg were solubilized and their localization on sperm was determined (Burkin and Miller, 2000) to be initially the apical ridge (the initial crescent on the sperm head to which the instant invention antibody-binding biomarker binds), followed by the entire acrosomal region (to which the antibody-binding biomarker binds as ejaculates age). Burkin and Miller conclude, “Based on these results, we expect to find primary zona receptor candidates on the apical ridge of acrosome-intact sperm. Secondary zona receptor candidates on acrosome-reacted sperm are expected to be found over the entire acrosomal region.”

The instant inventor has found that sperm bind the Fc biomarker, initially on the apical ridge of acrosome-intact sperm, then expanding to bind the entire acrosomal region, while the acrosome is associated with sperm. FIG. 28 shows the association of solubilized zona-binding proteins with the apical ridge of sperm (from Burkin and Miller, 2000). This is coincident with the initial site of labeling with the instant invention biomarker-based assay. Though not being bound by theory, the Inventor's antibody based Fc receptor biomarker functions endogenously in capacitated sperm as an egg-binding ligand.

Treatment to Control Sperm Status

The instant methods and observations as described herein provide a means to measure sperm capacitation status and adjust freezing or insemination accordingly, to optimize fertility or create gender bias. The instant methods and observations as described herein provide a means not just to respond to sperm status, but to control sperm status by addition of reagents or by other treatments. Many agents are known in the art to affect capacitation. When used in conjunction with the assays of the instant invention, these agents simplify or shorten processing while retaining desirable traits/performance of sperm. Sites of intervention in capacitation are found in FIG. 18. Non limiting examples of agents described in the art as slowing of capacitation are endocannibinoids (marijuana contains exogenous cannibinoids) Barboni and colleagues (2011). Other agents reported to affect capacitation include bicarbonate, bovine serum albumin and the non-hydrolyzable analog of cAMP, 8-buteryl cAMP. Reversibility, by bicarbonate and bovine serum albumin treatment, of early events in capacitation—calcium signaling, acrosomal reactivity, and protein phosphorylation—was also demonstrated by Bedu-Addo et al., 1995). A combination of one or more agents affecting sperm capacitation with the instant invention biomarker assay is useful in controlling capacitation, not just responding to it.

The instant invention also offers the potential of optimizing sperm even further for fertilization, through use of biomarkers that have been associated with good fertility but have never been optimized by a multipoint assay on sperm before fertilization. Such biomarkers include FAA (fertility associated antigen) of McCauley et al. (2004), adhesion markers measured on sperm used for in vitro fertilization (IVF)—including the VLA (very late antigen) integrins and fibronectins (Glander et al., 1996). While their presence on sperm was associated with improved fertility in single point assays for IVF fertilization, they have never been evaluated as targets in a multipoint assay prior to fertilization.

Some of these targets for controlling sperm capacitation are druggable. Use of the instant invention assays to screen for hits and eventually therapeutic agents could produce additional agents to control capacitation for use in conjunction with these assays.

FIG. 1 lists many assays exist for sperm maturation at the research, veterinary and clinic levels, but they have not been applied to fresh ejaculates as multi-point, real time assays to optimize an ejaculate's or a semen sample's fertility prior to insemination. Some specific citations are included, other potential capacitation probes are covered in the reviews already cited. Their use thus results in randomly-timed assessment of sperm maturation as a snapshot in time, not as a movie. This shortcoming compromises fertility optimization by: (1) failing to enable detection of the optimal degree of fertility and (2) therefore preventing stabilization of sperm at the optimal metabolic state. The instant inventor's methods overcome these shortcomings.

Processing

Once the results of the assessment of a specific performance(s), e.g., fertility and/or the generation of a sex bias are obtained, the semen is diluted directly or washed and diluted (extended) using commercially available products such as Bioxcell® (IMV, L'Aigle, France), or egg yolk based extenders, or the extenders Triladyl® and Andromed® (Minitube, Tiefenbach, Germany) before insemination or processing e.g., freezing and/or packaging into straws for use in insemination. Cryoprotectants suitable for use in freezing include, but are not limited to sugars such as trehalose, glycerol, propylene glycol, dimethylsulfoxide and egg yolk.

Kit

One embodiment comprises a kit for carrying out the disclosed assay. The kit contains at least a first binding protein or carbohydrate reactive with a biomarker, either directly or indirectly, and optionally a means for detecting said biomarker, and a description of use of the kit. In some embodiments the kit further comprises for example, a dye, or at least a first binding protein and optionally a second binding protein, and a sampling container and/or incubator. Further, in some embodiments, the kit contains instructions for observing morphological changes that reflect the metabolic changes used to predict field traits, for instance by brightfield micoscopy.

The methods described herein are designed for smooth integration into current methods of semen processing and, when properly implemented as described herein, create a desired performance, e.g., an increase in fertility and/or a sex bias in a time-based method. Preferably, this occurs under conditions that maintain fertility in standard on-site techniques of artificial insemination. The method preferably preserves the number of cells processed, because methods that cause a reduction in cell yield are unsuitable in terms of economic and fertility losses. All methods requiring a physical separation of sperm reduce cell yield.

The present methods preferably achieve increased fertility and/or creation of a gender bias by enabling detection of sperm metabolic changes that indicate when collected semen should be processed. As shown in FIG. 13, assay results enable users to process semen at a time that can increase fertility and/or cause sex bias, and/or have another desirable trait upon processing for use, e.g., artificial insemination (AI).

Also described herein is the application of the real time, multipoint assays for optimizing performance of sperm cells to cells other than sperm cells which are undergoing physiological changes over time. Non limiting examples of such cells include hybridoma cells or other fused cells, genetically transformed cells, and stem cells.

For example, the multipoint, real time assays described herein are applied to a culture of hybridoma cells in optimizing the performance of the antibodies from the culture upon harvesting. In preferred embodiments, the performance includes, but preferably is not limited to the therapeutic efficacy, half life, glycosylation status of antibodies produced by hybridoma cells in culture, and cell viability or resistance to processing of the hybridoma cells. These assays can be used to harvest hybridoma cultures at an appropriate time for obtaining, for example, antibodies of optimum therapeutic efficacy and/or optimum half life as reduced clearance in vivo is related to carbohydrate status of the antibodies.

In another example, the multipoint, real time assays described herein are applied to a culture of stem cells in optimizing the performance of the stem cells from the culture upon harvesting for use in transplantation procedures. In preferred embodiments, the performance includes, but preferably is not limited to the therapeutic efficacy, and includes cell viability or resistance to processing of the stem cells for transplantation. These assays can be used to harvest the stem cell culture at an appropriate time for obtaining, for example, stem cells of optimum therapeutic efficacy for transplant.

In another example, the multipoint, real time assays described herein are applied to a cultures of blood products in optimizing the performance of the blood product upon processing for use in in vitro or in vivo procedures. In preferred embodiments, the performance includes, but preferably is not limited to the therapeutic efficacy, and resistance to processing of the blood product for use in in vitro or in vivo procedures. These assays can also be used to ensure a particular blood product has a long shelf life.

In another example, the multipoint, real time assays described herein are applied to methods to identify druggable targets, on cells including sperm cells. Assays can also—Because the assays described herein are rapid, run in vitro and easy to automate—, the multipoint, real time assays described herein are used in drug development to screen libraries to identify hits directed to numerous targets, to obtain therapeutics that can improve fertility or act as contraceptives, or have other pharmacologically beneficial uses.

All patents, patent applications, and published references cited herein are hereby incorporated by reference in their entirety. The disclosure set forth herein has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope encompassed by the appended claims.

WORKING EXAMPLES Example 1 Annexin Treatment

The sperm from a 10 μl sample of ejaculate is washed twice with PBS buffer (8 g NaCl; 0.2 g KCl; 1.44 g Na₂HPO₄.7H₂O; 0.24 g KH₂PO₄; H₂O to 1 liter. pH 7.2) and the cells resuspended at a concentration of ˜1×10⁶ cells/ml in 1× Binding Buffer (10× Buffer is 0.1 M HEPES, pH 7.4; 1.4 M NaCl; 25 mM CaCl₂).

100 μl of the solution (−1×10⁵ cells) is transferred to a 5 ml culture tube and 2 μl of Annexin V stock solution (Annexin V-PE; BD Biosciences cat. no. 556422, 556421) added. The cells are gently mixed and incubated for 5 min. at room temperature in the dark. 400 μl of 1× Binding Buffer is added and the cells analyzed immediately by fluorescence microscopy to determine the number of cells positive for apoptosis (i.e. Annexin V positive).

Controls are (1) unstained cells, (2) experimental cells stained with Annexin V-FITC for 5 min. and (3) blocked cells stained with 5 mg of unlabeled Annexin V and then Annexin V-FITC. Annexin A5 is used as a probe to detect cells that have expressed phosphatidylserine on the cell surface, a feature found in apoptosis as well as other forms of cell death

Blocked Cell Control

This control includes preincubation of cell samples with recombinant unconjugated Annexin V, which is included as part of the BD Annexin V-FITC Apoptosis Detection Kit II (Cat. No 556570). This serves to block Annexin V-FITC binding sites and thus demonstrates the specificity of Annexin V-FITC staining.

The protocol is essentially as described above, but 5-15 μg of purified recombinant Annexin V is added instead of Annexin V-PE. The amount of purified recombinant Annexin V required to saturate binding sites may vary according to cell type, and stage of apoptosis. In some cases the cell number is reduced to 0.5×10⁵/100 μl, still adding 5-15 μg of recombinant Annexin V, to obtain optimal results. In addition, the resulting Annexin V mixture is incubated at room temperature for 15 minutes before adding 5 μl of Annexin V-FITC, mixing and incubating again at room temperature for 15 minutes in the dark. 400 μl of 1× Binding Buffer is added and the cells analyzed immediately by fluorescence microscopy.

Increased sex bias is produced, in the case of annexin V, using a jump point when 20-40% of sperm show annexin V positivity.

Example 2 Semen Preparation and Processing

A device according to PCT/US09/38134 was incubated at 32° C. for 60 minutes or more to ensure uniform heating of the device. Just prior to use, the device was removed and within 2 minutes of collection, the sperm was placed in the collection container, inverted once, then immediately placed at 12° C. for at least 15 minutes before sampling is begun. During the sampling time the collection of sperm is maintained at 12° C.

5 μl of undiluted semen is mixed in a 1.5 ml microfuge tube with 100 μl antibody diluent with Bovine Serum Albumin (Invitrogen SKU #00-3118). Twenty (20) μl of primary antibody (rabbit anti-Salmonellas; Salmonella H antiserum A-Z product number 224061; Difco, Detroit, Mich.; reconstituted according to the manufacturer's instructions) is added, followed by 5 μl of secondary antibody conjugated to Alexa Fluor® 488 (goat anti-rabbit IgG (H+L); Invitrogen, Carlsbad, Calif., catalogue no. A11008) (2 mg/l) and mixed. The solution is then incubated in the dark for 20-30 minutes at ambient temperature. In general, the ambient temperature ranged from 7° C.-27° C.

After incubation, 1 ml of PBS buffer (8 g NaCl; 0.2 g KCl; 1.44 g Na₂HPO₄. 7H₂O; 0.24 g KH₂PO₄; H₂O to 1 liter. pH 7.2) is added and the solution microfuged for 20 seconds before removing the supernatant. The cell pellet is gently resuspended in 100 μl of PBS buffer and approximately 5 μl transferred to a microscope slide. The number of sperm exhibiting green fluorescence on the head (deemed “positive”) is counted as well as the total number of cells. A minimum of 100 cells are counted and the “percent positive” (% positive) is determined by dividing the number of positive cells by the total number of cells and multiplying by 100.

In this case, the processing time is determined based on the % positive. If there are less than 25% positive, incubation is continued for approximately 1 hour before repeating the assay. If more than 25% positive, the assay is repeated at a shorter time interval such as 5, 10 or 15 minutes. Values of the % positive reach a peak and then begin to decline. Two hours after the peak, Bioxcell® extender or egg yolk-based phosphate buffer extender equilibrated to 12° C. is added to the semen, semen is cooled to 4° C.-10° C. and semen is processed into straws or used immediately. Any method can be used for processing into straws, including methods described in by P. Bermejo-Alayerez et al. (2008) Biol of Reproduction 79:594-597).

Example 3 Variability of Sperm Biology

Semen was collected from dairy bulls (time=0) and incubated. At intervals, samples were taken and assayed according to Example 2. (A) assay of collections on two different days from a Canadian Holstein bull named Bacardi. (B) assay of collections on two different days from an Irish Friesian bull named RDU. Results are presented in FIG. 5.

Variability of sperm cell biology between bulls and between collections is revealed by the large differences in assay curve shape and in the time required for attainment of the assay peak point.

Example 4 Fertility and Gender Bias Increases in Working Dairy Herds Correlates with Assay Peak Point

The first ejaculate of Freisian bull RDU was collected on two different days as shown in FIGS. 10 (A) and (B). On each day, half of the ejaculate was processed at 6 h. The other half of the ejaculate was processed according to Example 2. In FIG. 10 (A) bull RDU collection was processed into cohorts RDU-E (assay-based, 5 hour incubation) and RDU-F (fixed time, 6 hours). In FIG. 10B, bull RDU collection was processed into cohorts RDU-M (fixed time, 6 hours) and RDU-N (assay-based, 7 hours incubation). A fertility difference of about 10% was observed between the two processing methods, independent of whether the assay indicated processing time should occur before or after the fixed 6 h time point. Results for gender bias and fertility appear in Examples 5-7 below.

Example 5 Fertility Increase in Working Dairy Herd

The first ejaculate of elite dairy bulls was collected and sperm cells assayed according to a “fixed time” incubation process or according to Example 2. For fixed time incubation, 12° C. extender was added to the 12° C. incubated semen and processed into frozen straws 6 hours post-collection. For semen assayed according to Example 2, the incubated semen was extended and processed into straws 2 hours after the assay revealed a peak in the percentage of assay-positive sperm (% positive). The time at which this peak was obtained varied between collections.

Two bulls were collected on different days to obtain semen treated by incubation. The control treatment involved the same two bulls with the collections of semen being processed into frozen straws according to standard methods. Success was measured by the non-return rate (NRR) of cows and heifers. NRR is a measure of fertility and is the percentage of animals in the dairy herd that do not return for repeat insemination due to pregnancy or death. A high NRR correlates with high herd fertility and is desirable.

Table 1 presents the results obtained and reports the number of animals serviced through AI, the number of animals returned after 30 days due to failure to become pregnant and the percent of non-return rate (% NRR). The normal heat cycle of a cow is 21 days.

TABLE 1 Increased Fertility in Working Dairy Herd Incubation Treatment method #Serves 30 Day % NRR Control n/a 1010 158 84.36 Incubation Fixed time 164 38 76.83 Assay-based 139 18 87.05

From the results presented it is clear that sperm obtained from the assay and used for insemination produced a greater number of pregnancies; that is, as a population had increased fertility. For example, in the fixed time group, 164 animals were serviced by artificial insemination. By 30 days after initial service, 38 of these were returned for repeat insemination, as they were not pregnant. About 77% of the animals were therefore NOT returned for repeat insemination, giving a 76.8% non-return rate. In contrast, for the assay group, 139 animals were inseminated and only 18 were returned for repeat insemination by day 30, meaning 87% of the animals were not returned for repeat insemination.

In the control group, the non-return rate was 84.36% while in the assay group the NRR was 87.05, which indicates a fertility improvement of 2.69%. The fixed time group showed damage to fertility. Using logistic regression with pregnancy as the outcome, the improvement obtained by the assay method is statistically significantly more likely to increase fertility than the fixed time method (log odds ratio=0.71, p=0.047; a log odds ratio of 0 implies no association).

Example 6 Fertility Increase Requires Incubation Time Variability

In order to determine the effect of incubation on fertility, the first ejaculate of two elite dairy bulls, RDU and QUR was collected. Each semen collection from each bull was divided in half. One half of the collection was processed according to a “fixed time” incubation process and one half according to Example 2. For the fixed time incubation, 12° C. extender was added to the 12° C. incubated semen and processed into frozen straws 6 hours post-collection. For semen assayed according to Example 2, the incubated semen was extended and processed into straws 2 hours after the assay revealed a peak in the percentage of assay-positive sperm (% positive).

Table 2 presents the data obtained and reports the number of animals serviced through AI, the number of animals returned after 30 days due to failure to become pregnant (NNR) and the percent of non-return rate (% NRR). The normal heat cycle of a cow is 21 days.

TABLE 2A Fertility Increase Is Correlated With Assay Collection Pair 30 and Bull Time, h Method #Serves Day % NRR RDU 6 Fixed 51 11 78 E&F 5 Assay 74 8 89 QUR 6 Fixed 8 2 75 I&J 7 Assay 20 3 85 RDU 6 Fixed 100 25 75 M&N 7 Assay 39 6 85 The data in Table 2A indicates that the quality of the sperm obtained from the assay method results in more pregnancies and a lower % NRR. This increase in fertility shown in Table 2A is consistent with an increase in fertility as evidenced by a reduced non return rate from a field study on dairy farms in which ejaculates were split into two portions, one portion being prepared for insemination at a time point determined according to the antibody based FC receptor biomarker method described herein with 30 minute time intervals. The second portion of each ejaculate was prepared for insemination at a fixed time point. The data in Table 2B indicates that the quality of the sperm obtained from the assay method of the invention results in more pregnancies and a lower % NRR than the conventional method.

TABLE 2B Production of statistically significant (p = 0.10) increased fertility by the biomarker-half of 5 split ejaculates compared to a control ejaculate half that was also incubated, but not according to biomarker assay timing, on dairy farms. Control incubation treatment involved dilution and freezing of the ejaculate 1 h before or 1 h after the optimal time indicated by biomarker assay. Split Non-return ejaculate # Cows for rate (measure of incubation # repeat AI (not pregnancy, thus % treatment Inseminations pregnant) fertility) increase Biomarker 400 108 73.0% 6.7% Control 329 104 68.4% —

Example 7 Gender Bias in Working Dairy Herd

In order to determine the effect of incubation on gender bias, the first ejaculate of elite dairy bull RDU was collected as described in Example 3. Each semen collection was divided in half. One half of the collection was processed according to a “fixed time” incubation process and one half according to Example 2. For the fixed time incubation, 12° C. extender was added to the 12° C. incubated semen and processed into frozen straws 6 hours post-collection. For semen assayed according to Example 2, the incubated semen was extended and processed into straws at the time shown in Table 3 (e.g. 2 hours after the assay revealed a peak in the percentage of assay-positive sperm (% positive).

Table 3 presents the data obtained and reports the numbers of each gender produced as determined by fetal scanning.

TABLE 3 Gender bias according to fetal scanning Collection Cohorts and # # % Bull Time, h Method Females Males Female RDU- 5 Assay-based 15 9 63 E&F 6 Fixed 10 8 56 RDU- 6 Fixed 24 17 59 M&N 7 Assay-based 10 5 67

As can be seen from the results, the assay-based treated semen in accord with the present invention produced more females, as desired, than the fixed time treatment.

Table 4 summarizes the results obtained when the data from Table 3 is grouped according to fixed-time or assay-based treatment.

TABLE 4 Summary of gender bias based on semen treatment Incubation % Treatment method # Females # Males Female* Control n/a n/a n/a 50 Incubation Fixed time 34 25 58 Assay-based 25 14 64 *for control, historical percentage of females is reported

Example 8 Semen Preparation and Processing—Enzymatic

A device according to PCT/US09/38134 is incubated at 32° C. for 60 minutes or more to ensure uniform heating of the device. Just prior to use, the device is removed and within 2 minutes of collection, the sperm is placed in the collection container, inverted once, then immediately placed at 12° C. for at least 15 minutes before sampling is begun. During the sampling time the collection of sperm is maintained at 12° C.

20 μl of undiluted semen is mixed in a 1.5 ml microfuge tube with 1000 μl antibody diluent with Bovine Serum Albumin (Invitrogen SKU #00-3118). Five (5) μl of primary antibody (e.g. rabbit anti-Salmonella; Salmonella H antiserum A-Z product number 224061; Difco, Detroit, Mich.; reconstituted according to the manufacturer's instructions) is added, mixed and incubated in the dark for 15 minutes at ambient temperature. 10 μl as supplied by the manufacturer of a peroxidase-conjugated goat anti-rabbit IgG (H+L) (catalogue no. Invitrogen SKU # G-21234) secondary antibody is added.

After incubation, 1 ml of PBS buffer (8 g NaCl; 0.2 g KCl; 1.44 g Na₂HPO₄. 7H₂O; 0.24 g KH₂PO₄; H₂O to 1 liter. pH 7.2) is added, the resulting solution mixed gently and centrifuged. The supernatant decanted. This wash step is repeated five times.

To produce signal, 1 ml of peroxidase soluble substrate (TMB/E Ultra Sensitive, Blue, Horseradish Peroxidase Substrate (soluble); Millipore) is added, mixed gently and incubated at ambient temperature for 10 minutes.

The tube is placed in a calibrated colorimeter and the absorbance reading taken.

Proportionally smaller volumes can be used to accommodate performing the assay in a 96 well format or other configurations.

The processing time is determined based on the optical density (OD) reading. Incubation is continued for approximately 1 hour before repeating the assay. Once the OD reading has increased by about 20% above background, the assay may be repeated at a shorter time interval such as 5, 10 or 15 minutes. With time, the OD reading will again decline. Two hours after the peak OD reading, Bioxcell® extender or egg yolk-based phosphate buffer extender equilibrated to 12° C. is added to the semen, semen is cooled to 4° C.-10° C. and semen is processed into straws or used immediately.

Example 9 Gender Bias from In Vitro Fertilization Studies

Semen was collected from an elite diary bull, KSY, and was either placed into a jacketed collection tube pre-warmed to 32° C., which was then placed and held at 12° C. (i.e. control), or processed as described in Example 2.

In vivo analysis of fixed time and assay-based artificial insemination of working herds was conducted. Based on fetal screening, the assay-based method gave higher gender bias than the fixed-time method. In contrast, during in vitro analysis of single fixed time and assay-based incubations, the fixed incubations gave higher gender bias than assay-based. In both cases, however, the percent female embryos is elevated above baseline by in vitro analysis. Table 5 presents the results of in vitro analysis.

TABLE 5 Gender bias detected by in vitro fertilization of bovine eggs, followed by PCR-based gender analysis. Male Female Not sexed Treatment n (%) n (%) n (%) Control 77 (59.2) 53 (40.8) 1 (0.7) Assay-based 77 (57.9) 56 (42.1) 1 (0.7) Fixed time 60 (51.3) 57 (48.7) 2 (1.7) Total 214 (56.3)  166 (43.7)  4 (1)  

Example 10 Gender Bias Produced in Field Trials

Nine ejaculates from 5 bulls were processed by two variants of the method in accordance with the present invention. Cows and heifers were serviced by standard Art Insemination. Fetal Scanning was performed to evaluate calf gender. National Irish Cattle Breeding Federation (ICBF) records were used for controls.

The Field Trial results are shown below.

TABLE 6 Gender Bias is Produced and is Statistically Significant # Male Percentage Treatment Total Births # Female Births Births Female Control 36,674 18,443 18,231 50% Methods 498 285 213 57% P = 0.001 Described herein

A statistical analysis of these results indicates that the null hypothesis that the gender bias in the experimental group is equivalent to the gender bias in the control group can be rejected with a two-sided P-value of 0.001 from a chi-square test of equality of proportions. The confidence interval means that, with probability of 95%, the true gender bias is between 53% and 62%.

Example 11 Male or Female Bias from Aliquots of the Same Ejaculate Using Biomarker-Based Processing of Semen into Frozen Doses Achieved by Changing Time of Cell Stabilization by Dilution, Cooling and Freezing, Relative to Biomarker Assay Result

Collections were split and processed (to stabilize cells, by addition of diluent and immediate transfer to 4 C for cooling prior to freezing) at 5 h (for male) or 6 h (for female) after biomarker either showed a peak—of maximum % positive cells—rising from baseline, or the first increase of the peak that was rising from baseline (if the rising part of the peak contained at least two points). Semen doses prepared by these two methods were used to AI cows and heifers in working dairy herds with frozen/thawed doses at standard 15 million cells/0.25 ml straw in BioXcell extender. Calf sex was determined at birth with a calving interval of +/−10 d. Usual female births=48.6% (Berry et al., 2011).

The results show a significant increase in male newborns (55%), see Table 7.

Male or Female Bias from Biomarker-based Processing of Semen into Frozen Doses, Achieved by changing time of cell stabilization by dilution, cooling and freezing, relative to biomarker assay result Male bias processing (dilution, cooling Female bias processing (dilution, cooling and freezing of doses 2 h post-max % and freezing of doses 2 h post-max % biomarker positive sperm) biomarker positive sperm) Split Bull & # # % Bull & # # % Ejaculate XT Total n Females Males Male XT Total n Females Males Female RDU RDU M 79 36 43 RDU N, 31 16 15 M & N 6 h 7 h QUR QUR I 20 9 11 QUR J, 52 29 23 I & J 6 h 7 h RDU RDU O 11 5 6 RDU P, 13 8 5 O & P 6 h 7 h TOTAL 110 50 60 55% 96 53 43 55% Male Female (7% (13% increase) increase)

Example 12 Small Field Trial Results—Split Ejaculates Demonstrate Male Bias

Collections were split and processed by the indicated method and used to AI cows and heifers in working dairy herds with frozen/thawed doses at standard 15 million cells/0.25 ml straw in BioXcell extender. Calf sex was determined at birth with a calving interval of +/−10 d. Usual female births=48.6% (Berry et al., 2011).

Method 2 (time) Method 2 + Cohen Biomarker (EnGender) Split Bull & # # % Bull & # # % Ejaculate XT Total n Females Males Female XT Total n Females Males Female RDU RDU E 39 17 22 RDU 34 21 13 E & F 6 h F, 6 h RDU RDU M 79 36 43 RDU N, 31 16 15 M & N 6 h 7 h QUR QUR I 20 9 11 QUR J, 52 29 23 I & J 6 h 7 h RDU RDU O 11 5 6 RDU P, 13 8 5 O & P 6 h 7 h RDU RDU T 16 5 11 RDU 18 12 6 S & T 7 h S, 6.5 h TOTAL 165 72 93 44% 148 86 62 58%*

Example 13 Fertility is Maintained in the Present Methods Producing Gender Bias Produced in Field Trials, but not by Method Measuring Elapsed Time

Ten collections of semen were obtained from 5 bulls and processed by either control or a method of the present invention. (One bull—one collection—was later eliminated due to excessive broken heads, and one for abnormal assay curve). Cows and heifers were serviced by standard Artificial Insemination. National Irish Cattle Breeding Federation (ICBF) records were used for NRR.

TABLE 8 Control Fertility and Fertility Generated by the methods described herein are Statistically Indistinguishable Treatment Total Births 60 Day NRR 60 Day % Control Methods 1,469 335 0.7583 Methods 1,469 388 0.7359 described herein Statistical analysis of the field trial results above indicates that using a two-sided, 0.05 level Fisher's exact test, the control and experimental cohorts are not significantly different (p=0.174).

Example 14 The Cohen Fertility Assay—Clinical (CFAC) Standard Operating Procedure (SOP) v. 1.0 (March 2012) Clinical Multipoint Fertility Optimization 1.0 Intended Use

The Cohen Fertility Assay (CFAC) is designed for use in process control during preparation of semen for insemination in clinical or veterinary applications.

2.0 Introduction

In conventional semen processing, semen is collected, cooled assayed once and prepared for insemination. In CFAC processing, semen is collected, cooled, assayed at multiple time points by the CFAC method. Semen processing is adjusted based on assay data to optimize fertility at insemination.

3.0 Principle

Sperm undergo a maturation process upon ejaculation. Sperm mature in groups, with fully activated sperm having a short life during which they will fertilize an egg, or they will die. CFAC detects the presence a biomarker that appears during sperm maturation. This marker can be used to determine the maturational status of a group of sperm. By knowing the maturational status of a sperm group, it is possible to capture them in an optimally fertile state, and thus enhance male-side fertility.

4.0 Reagents and Materials

The CFAC is supplied as reagents pre-measured into single use tubes. Standard laboratory supplies are required but not provided. A sample collection cup is required but not provided. Scoring of the CFAC is by cytometry.

5.0 Safety and Handling Precautions

5.1 Safety Precautions

Follow standard safety procedures for handling semen as OPIM (other potentially infectious material). Do not pipette by mouth. Do not smoke, eat or drink in areas in which samples or kit reagents are handled. Wash hands before leaving laboratory. Clean spills promptly. Dispose of materials properly. Sodium azide may react with lead or copper piping to form highly explosive metal azides. When disposing of liquid waste, dilute thoroughly to prevent the formation of such products.

5.2 Handling Precautions

Do not use kit components beyond their expiry date. Comply with instructions.

6.0 Sample Collection

The assay is performed directly on 10 ul aliquots of raw semen. Semen is collected into the collection cup and the cup is handled as described on the SOP.

Example 13 CFAC Collection Cup Procedure Standard Operating Procedure Safety

Follow standard procedures for handling of semen.

-   -   1. INSPECT COLLECTION CUP         -   a. Visually inspect device for cracks or damage before             using. Use only devices that are intact.     -   2. BRING CUP INSULATOR TO OPERATING TEMPERATURE         -   a. Place Cup Insulator in 32° C. water bath for at least 60             minutes. Cup Insulators may be left in bath overnight for             use the next day.     -   3. PERFORM COLLECTION AND BEGIN INCUBATION         -   a. Obtain semen sample in collection cup.         -   b. As soon as possible, and within 3 minutes of ejaculation,             place Collection Cup into Cup Insulator and place Collection             Cup Insulator, containing Collection Cup, into 12° C. water             bath.         -   c. Measure semen parameters by removing a sample for             analysis.     -   4. COHEN FERTILITY ASSAY         -   Follow instructions in the Cohen Fertility Assay SOP for             evaluating cells and carrying out further processing.

7.0 Assay Procedure

-   -   7.1 Equipment Required         Precision micropipettes or similar equipment with disposable         tips for measurement of 10 ul and 1 ml are required. Calibration         of these pipettes must be checked regularly. A calibrated         cytometer is needed. Also needed are a microfuge capable of         accepting 1.5 ml snap-cap tubes, 1.5 ml microcentrifuge tubes,         and distilled water.

Example 14 Cohen Fertility Assay—Clinical Standard Operating Procedure Safety

Follow standard procedures for handling of semen. Before running this assay, be sure that semen is collected incubated exactly as instructed in the CFAV Collection Cup SOP.

-   -   TREAT         -   i. Remove one 1.5 ml tube of pre-measured CFAV reagent from             kit         -   ii. Measure 10 ul of semen with a micropipette         -   iii. Add 10 ul of semen to CFAV reagent tube         -   iv. Mix well     -   INCUBATE         -   d. Place tube in dark for 20 minutes at ambient temperature     -   WASH         -   e. Add 1 ml BUFFER at ambient temperature         -   f. Microfuge 1 min         -   g. Carefully remove supernatant with 1 ml pipet.     -   10. SCORE         -   a. Place aliquot of resuspended cells into cytometer SIP             tube and analyze on a calibrated cytometer using the “CFAC             Assay” template (see Assay Scoring SOP for further details).     -   11. DETERMINE PROCESSING TIME         -   c. Plot percentage of positive cells. The percentage of             cells that are positive will usually peak in the timeframe             of 30 min-4 h post-collection. Time zero=time of collection.         -   d. 1.5 h after the peak positivity of cells for the CFAC             Biomarker, semen should be stabilized by cooling and             dilution with cryoprotectant if desired. Caution: deviation             from 1.5 h can cause process failure! Extend semen and             process semen in the standard protocol in use at your             clinic, with the following change: use extender cooled to             12° C. before it is added.

Example 15 CFAC Assay Scoring—with Accuri C6 Cytometer Standard Operating Procedure Safety

Follow standard procedures for handling of semen. Before scoring this assay, be sure that semen is collected and incubated exactly as instructed and that the assay is run exactly as instructed, to minimize process failures.

-   -   1. START EQUIPMENT         -   i. Turn on computer         -   ii. Turn on cytometer (Accuri [Becton Dickenson], Lansing,             Mich.)         -   iii. If needed, empty waste bottle and fill sheath bottle             with DI water     -   2. OPEN TEMPLATE AND NAME FILE         -   a. Click CFAC Assay Template         -   b. Select File>Save CFlow file as . . .         -   c. Name file by date by typing date code under File Name         -   d. Click Save     -   3. COLLECT DATA         -   a. Under the red Collect tab, click on desired cytometer             grid (1A is for first sample, first patient, 1B is for             second sample, first patient. 2A is for first sample, second             patient, etc.)         -   b. Next to cytometer grid (e.g., A01) type sample             information.         -   c. Check that stoplight shows green color. Load sample onto             SIP tube on cytometer and pull out plastic tube support             underneath tube         -   d. Click Run         -   e. After data are acquired, adjust gate on density plot so             that the percentage of positive sperm (the population on the             right) can be determined. Record this number. Remove sample             from SIP tube         -   f. For the next sample, repeat process above starting with             step 3a         -   g. After the samples for that hour are finished, click             Backflush, wait for stoplight to show green, then click             Unclog     -   4. CLEAN AND SHUT DOWN EQUIPMENT         -   a. Between every sample, place towel under SIP, select             Backflush or Unclog         -   b. At the end of the day, place a tube of cleaning fluid on             the cytometer, select well H11 and click Run. Run for 2             minutes. Then place a tube of water on the cytometer, select             well H11 and click Run. Run for 2 minutes. Allow water to             clean system for at least 2 minutes.         -   c. Turn off cytometer, then turn off computer     -   5. Maintenance—follow instructions in Accuri manual for         instrument cleaning. This should include every Wednesday and         Friday at day's end the extended cleaning of flow cell (found         under the Instrument tab on the cytometer menu).

8.0 Quality Control

A release QC test, designed for use on CFAC frozen semen doses, is under development.

9. Results

To date, results with animal studies indicate that the process is capable of a 4-5% increase in fertility.

10. Limitations

Assay reagents with cloudy or otherwise abnormal appearance should not be used.

Example 16 The Cohen Fertility Assay—Veterinary (CFAV) Standard Operating Procedure (SOP) v. 1.0 (March 2012) Veterinary Multipoint Fertility Optimization 1.0 Intended Use

The Cohen Fertility Assay—Veterinary (CFAV) is designed for use in process control during semen manufacture for frozen doses of veterinary semen.

2.0 Introduction

In conventional semen processing, semen is collected, cooled and extended for packaging into straws for freezing. In CFAV processing, semen is collected, cooled, assayed by the CFAV assay, extended and frozen at a time that will enable optimal fertility upon AI of cows and heifers using standard techniques.

3.0 Principle

Sperm undergo a maturation process upon ejaculation. Sperm mature in groups, with fully activated sperm having a short life during which they will fertilize an egg, or they will die. CFAV detects the presence a biomarker that appears during sperm maturation. This marker can be used to determine the maturational status of a group of sperm. By knowing the maturational status of a sperm group, it is possible to capture them in an optimally fertile state, and thus enhance male-side fertility.

4.0 Reagents and Materials

A collection tube, the CFAV device, and an SOP for use is supplied. The CFAV assay reagents are supplied as three color-coded tubes (Green 1, Red 2 and Blue 3) plus a wash buffer (Buffer), and this SOP for use. Standard laboratory supplies are required. Scoring of the assay is by cytometry.

5.0 Safety and Handling Precautions

5.1 Safety Precautions

Follow standard safety procedures for handling livestock and cattle semen. Do not pipette by mouth. Do not smoke, eat or drink in areas in which samples or kit reagents are handled. Wash hands before leaving laboratory. Clean spills promptly. Dispose of materials properly. Sodium azide may react with lead or copper piping to form highly explosive metal azides. When disposing of liquid waste, dilute thoroughly to prevent the formation of such products.

5.2 Handling Precautions

Do not use kit components beyond their expiry date. Comply with instructions.

6.0 Sample Collection

The assay is performed directly on 5 ul aliquots of raw semen. Semen is collected into the pre-warmed CFAV collection device by attaching the device to the artificial vagina (AV). Standard Operating Procedure (SOP) for the CFAV device follows.

Example 17 CFAV Device Standard Operating Procedure Safety

Follow standard procedures for handling of semen.

-   -   5. INSPECT CFAV DEVICE AND INSERT BEAD         -   a. Visually inspect device for cracks or damage before             using. Use only devices that are intact.         -   b. Place one bead into each intact device.     -   6. BRING CFAV DEVICE TO OPERATING TEMPERATURE         -   a. Place device in 32° C. water bath for at least 60             minutes. Make sure device is submerged in water up to the             cap of the large tube, so the device warms uniformly.             Devices may be left in bath overnight for use the next day.     -   7. PERFORM COLLECTION AND BEGIN INCUBATION         -   a. Use standard methods of attachment to AV and of             collection. If device is out of water bath for more than 5             minutes between placement onto AV and collection, remove it             from AV and replace with another device from the 32° C.             water bath, so that the collection temperature will remain             near 32° C.         -   b. Within 1 minute of collection, retrieve device, cap and             invert once, then place immediately into 12° C. water bath.         -   c. Measure volume after device has been in the 12° C. bath             for at least 15 minutes, in order to minimize temperature             changes. Keep device submerged in water up to the cap of the             large tube during the cooling period to ensure a smooth and             uniform drop in the temperature of the ejaculate.     -   8. ASSAY         -   Follow instructions in the CFAV Assay SOP for evaluating             cells and carrying out further processing.

7.0 Assay Procedure

7.1 equipment required

Precision micropipettes or similar equipment with disposable tips for measurement of 5 ul, 10 ul, 20 ul, 100 ul and 1 ml are required. Calibration of these pipettes must be checked regularly. A calibrated cytometer is needed. Also needed are a microfuge capable of accepting 1.5 ml snap-cap tubes, 1.5 ml microcentrifuge tubes, and distilled water.

CFAV Assay Standard Operating Procedure Safety

Follow standard procedures for handling of semen. If carrying out microscopy, do not look directly into the fluorescent light. Before running this assay, be sure that semen is collected and incubated exactly as instructed in the CFAV DEVICE SOP to minimize process failures.

-   -   12. TREAT         -   i. Into 1.5 ml tube, pipet the following IMMEDIATELY before             use:         -   ii. 100 ul GREEN 1         -   iii. 20 ul RED 2         -   iv. 5 ul BLUE 3         -   v. 5 ul neat semen, mix.         -   vi. Assay at 30 min intervals. Keep reagents cool at all             times.     -   13. INCUBATE         -   a. Place tube in dark for 20 minutes at ambient temperature     -   14. WASH         -   a. Add 1 ml BUFFER at ambient temperature         -   b. Microfuge 30 seconds         -   c. Carefully remove supernatant with 1 ml pipet.     -   15. SCORE         -   a. Place aliquot of resuspended cells into cytometer tube             and analyze on a calibrated cytometer using the “CFAV Assay”             template (see Assay Scoring SOP for further details).     -   16. DETERMINE PROCESSING TIME         -   e. Plot percentage of positive cells. The percentage of             cells that are positive will usually peak in the timeframe             of 30 min-4-h post-collection. Time zero=time of collection.             1.5 h after the peak positivity of cells for the CFAV             Biomarker, semen should be extended, cooled further, and             then frozen. Caution: deviation from 1.5 h can cause process             failure! Extend semen and process semen in the standard             protocol in use at your AI station, with the following             change: use extender cooled to 12° C. before it is added.             CFAV Assay Scoring—with Accuri C6 Cytometer

Standard Operating Procedure Safety

Follow standard procedures for handling of semen. Before scoring this assay, be sure that semen is collected and incubated exactly as instructed and that the assay is run exactly as instructed, to minimize process failures.

-   -   6. START EQUIPMENT         -   i. Turn on computer         -   ii. Turn on cytometer (Accuri, Lansing, Mich.)         -   iii. If needed, empty waste bottle and fill sheath bottle             with DI water     -   7. OPEN TEMPLATE AND NAME FILE         -   a. Click CFAV Assay Template         -   b. Select File>Save CFlow file as . . .         -   c. Name file by date by typing date code under File Name         -   d. Click Save     -   8. COLLECT DATA         -   a. Under the red Collect tab, click on desired cytometer             grid (1A is for first sample, first bull, 1B is for second             sample, first bull. 2A is for first sample, second bull,             etc.)         -   b. Next to cytometer grid (e.g., A01) type sample             information and batch time (time when all collections are             batched for assay) For example, this would be written as RDU             9.30 for first time point from bull RDU)         -   c. Check that stoplight shows green color. Load sample onto             SIP tube on cytometer and pull out plastic tube support             underneath tube         -   d. Click Run         -   e. After data are acquired, adjust gate on density plot so             that the percentage of positive sperm (the population on the             right) can be determined. Record this number. Remove sample             from SIP tube         -   f. For the next sample, repeat process above starting with             step 3a         -   g. After the samples for that hour are finished, click             Backflush, wait for stoplight to show green, then click             Unclog     -   9. CLEAN AND SHUT DOWN EQUIPMENT         -   a. Between every sample, place towel under SIP, select             Backflush or Unclog         -   b. At the end of the day, place a tube of cleaning fluid on             the cytometer, select well H11 and click Run. Run for 2             minutes. Then place a tube of water on the cytometer, select             well H11 and click Run. Run for 2 minutes. Allow water to             clean system for at least 2 minutes.         -   c. Turn off cytometer, then turn off computer     -   10. Maintenance—follow instructions in Accuri manual for         instrument cleaning. This should include every Wednesday and         Friday at day's end the extended cleaning of flow cell (found         under the Instrument tab on the cytometer menu).

8.0 Quality Control

A release QC test, designed for use on CFAV straws after thawing, is under development.

9. Results

To date, results with EnGender indicate that the process is capable of production of a moderate female bias with preservation of fertility.

10. Limitations

Collections displaying abnormal quality at collection should not be processed with CFAV. Abnormal morphology, excessively thin or otherwise abnormal ejaculates should not be used. Assay reagents with cloudy or otherwise abnormal appearance should not be used.

Example 18 Veterinary Gender Bias Process Standard Operating Procedure v. 1.8 (July 2011) EnGender Process Control for Fertility-preserving Gender Bias 1.0 Intended Use

EnGender is designed for use in process control during semen manufacture.

2.0 Introduction

In conventional semen processing, semen is collected, cooled and extended for packaging into straws. In EnGender processing, semen is collected, cooled, assayed by the EnGender assay, and extended at a time that will enable production of a moderate female bias with good fertility upon AI of cows and heifers using standard techniques.

3.0 Principle

Sperm undergo a maturation process upon ejaculation. Sperm mature in groups, with fully activated sperm having a short life during which they will fertilize an egg, or they will die. This assay detects the presence of a biomarker, a marker that appears during sperm maturation. This marker can be used to determine the maturational status of a group of sperm. Knowledge of the maturation status of sperm is combined with another set of observations: (1) Y-bearing sperm, that fertilize to create males, mature and grow old slightly faster than X-bearing sperm and (2) X-sperm in a maturational group persist longer than Y-sperm. By knowing the maturational status of a sperm group, and that the earliest-maturing are Y-enriched while the longest-lasting are X-enriched, it is possible to time the cooling period to biologically enrich straws for X-bearing sperm, without a physical separation or sort of any type. EnGender therefore is a gentle treatment of sperm, producing moderate bias through use of natural mechanisms that already exist in nature. This preserves sperm integrity and enables AI of both heifers and cows under conditions that preserve fertility.

4.0 Reagents and Materials

A collection tube device, and an SOP for use is supplied. The biomarker assay reagents are supplied as three color-coded tubes (Green 1, Red 2 and Blue 3) plus a wash buffer (Buffer), and an SOP for use. Standard laboratory supplies are required. Scoring of the assay is by microscopy or cytometry. Cytomety is preferable and is the only scoring platform supported by for commercial use. Microscopy is supported solely for laboratory research.

5.0 Safety and Handling Precautions

5.1 Safety Precautions

Follow standard safety procedures for handling livestock and cattle semen. Do not pipette by mouth. Do not smoke, eat or drink in areas in which samples or kit reagents are handled. Wash hands before leaving laboratory. Clean spills promptly. Dispose of materials properly. Sodium azide may react with lead or copper piping to form highly explosive metal azides. When disposing of liquid waste, dilute thoroughly to prevent the formation of such products.

5.2 Handling Precautions

Do not use kit components beyond their expiry date. Comply with instructions.

6.0 Sample Collection

The assay is performed directly on 5 ul aliquots of raw semen. Semen is collected into the pre-warmed collection device by attaching the device to the artificial vagina (AV). Standard Operating Procedure (SOP) for the device follows.

Device Standard Operating Procedure Process Control for Fertility—Preserving Gender Bias Safety

Follow standard procedures for handling of semen.

-   -   9. INSPECT DEVICE AND INSERT BEAD         -   a. Visually inspect device for cracks or damage before             using. Use only devices that are intact.         -   b. Place one bead into each intact device.     -   10. BRING DEVICE TO OPERATING TEMPERATURE         -   a. Place device in 32° C. water bath for at least 60             minutes. Make sure device is submerged in water up to the             cap of the large tube, so the device warms uniformly.             Devices may be left in bath overnight for use the next day.     -   11. PERFORM COLLECTION AND BEGIN INCUBATION         -   a. Use standard methods of attachment to AV and of             collection. If device is out of water bath for more than 5             minutes between placement onto AV and collection, remove it             from AV and replace with another device from the 32° C.             water bath, so that the collection temperature will remain             near 32° C.         -   b. Within 1 minute of collection, retrieve device, cap and             invert once, then place immediately into 12° C. water bath.             Failure to carry out these steps quickly may result in             process failure.         -   c. Measure volume after tube has been in the 12° C. bath for             at least 15 minutes, in order to minimize temperature             changes. Keep tube submerged in water up to the cap of the             large tube during the cooling period to ensure a smooth and             uniform drop in the temperature of the ejaculate.     -   12. ASSAY         -   Follow instructions in the biomarker Assay SOP for             evaluating cells and carrying out further processing.

7.0 Assay Procedure

7.1 Equipment required

Precision micropipettes or similar equipment with disposable tips for measurement of 5 ul, 10 ul, 20 ul, 100 ul and 1 ml are required. Calibration of these pipettes must be checked regularly. A calibrated cytometer or fluorescence microscope (research use only) suitable for use with the fluorophore fluorescein isothiocyanate (FITC) is needed. Also needed are a microfuge capable of accepting 1.5 ml snap-cap tubes, such tubes, and distilled water.

Biomarker Assay Standard Operating Procedure Process Control for Fertility—Preserving Gender Bias Safety

Follow standard procedures for handling of semen. If carrying out microscopy, do not look directly into the fluorescent light.

Before running this assay, be sure that semen is collected incubated exactly as instructed in the DEVICE SOP to minimize process failures.

-   -   17. TREAT         -   i. Into 1.5 ml tube, pipet the following IMMEDIATELY before             use:         -   ii. 100 ul GREEN 1         -   iii. 20 ul RED 2         -   iv. 5 ul BLUE 3         -   v. 5 ul neat semen, mix.         -   vi. Assay at 30 min intervals. Keep reagents cool at all             times.     -   18. INCUBATE         -   a. Place tube in dark for 20 minutes at ambient temperature     -   19. WASH         -   a. Add 1 ml BUFFER at ambient temperature         -   b. Microfuge 30 seconds         -   c. Carefully remove supernatant with 1 ml pipet.     -   20. SCORE         -   a. Add ˜200 ul BUFFER to cell pellet and mix gently to             resuspend         -   b. FOR MICROSCOPE: Transfer ˜5 ul to slide and score #             positive sperm (green fluorescence on head) and #total             cells. Count at least 100 cells. Calculate % positive. (%             Positive=[# positive/# total cells]×100)         -   c. FOR CYTOMETER: place aliquot of resuspended cells into             cytometer tube and analyze on a calibrated cytometer using             the “Assay” template (see Assay Scoring SOP for further             details).     -   21. DETERMINE PROCESSING TIME         -   f. Plot percentage of positive cells. The percentage of             cells positive for the Biomarker will usually peak in the             timeframe of 30 min-4 h post-collection. Time zero=time of             collection.             Two hours (2 h) after the peak positivity of cells for the             Biomarker, semen should be extended. Caution: deviation from             2 h can cause process failure! Extend semen and process             semen in the standard protocol in use at your AI station,             with the following change: use extender cooled to 12° C.             before it is added.             Biomarker Assay Scoring—with Accuri C6 Cytometer

Standard Operating Procedure Process Control for Fertility—Preserving Gender Bias Safety

Follow standard procedures for handling of semen. Before scoring this assay, be sure that semen is collected and incubated exactly as instructed and that the assay is run exactly as instructed, to minimize process failures.

-   -   11. START EQUIPMENT         -   i. Turn on computer         -   ii. Turn on cytometer (Accuri, Lansing, Mich.)         -   iii. If needed, empty waste bottle and fill sheath bottle             with DI water     -   12. OPEN TEMPLATE AND NAME FILE         -   a. Click biomarker Template         -   b. Select File>Save CFlow file as . . .         -   c. Name file by date by typing date code under File Name         -   d. Click Save     -   13. COLLECT DATA         -   a. Under the red Collect tab, click on desired cytometer             grid (1A is for first sample, first bull, 1B is for second             sample, first bull. 2A is for first sample, second bull,             etc.)         -   b. Next to cytometer grid (e.g., A01) type sample             information and batch time (time when all collections are             batched for assay) For example, this would be written as RDU             9.30 for first time point from bull RDU)         -   c. Check that stoplight shows green color. Load sample onto             SIP tube on cytometer and pull out plastic tube support             underneath tube         -   d. Click Run         -   e. After data are acquired, adjust gate on density plot so             that the percentage of positive sperm (the population on the             right) can be determined. Record this number. Remove sample             from SIP tube         -   f. For the next sample, repeat process above starting with             step 3a         -   g. After the samples for that hour are finished, click             Backflush, wait for stoplight to show green, then click             Unclog     -   14. CLEAN AND SHUT DOWN EQUIPMENT         -   a. Between every sample, place towel under SIP, select             Backflush or Unclog         -   b. At the end of the day, place a tube of cleaning fluid on             the cytometer, select well H11 and click Run. Run for 2             minutes. Then place a tube of water on the cytometer, select             well H11 and click Run. Run for 2 minutes. Allow water to             clean system for at least 2 minutes.         -   c. Turn off cytometer, then turn off computer     -   15. Maintenance—follow instructions in Accuri manual for         instrument cleaning. This should include every Wednesday and         Friday at day's end the extended cleaning of flow cell (found         under the Instrument tab on the cytometer menu).

8.0 Quality Control

A release QC test, designed for use on biomarker-processed straws after thawing, is under development. The QC test will be used to confirm that the biomarker process has generated straws of semen enriched for viable X-sperm from the tested collections. We welcome inquiries about QC release test availability and can provide referrals to certified QC service laboratories.

9. Results

To date, results with biomarker indicate that the process is capable of production of a moderate female bias with preservation of fertility. Male bias and fertility improvements have also been noted, with protocol changes for male (1 h not 2 h wait period between biomarker signal and further processing by cooling, dilution, etc.) Fertility improvement can occur with this process but is not likely to be maximal.

10. Limitations

Collections displaying abnormal quality at collection should not be processed. Abnormal morphology, excessively thin or otherwise abnormal ejaculates should not be used. Assay reagents with cloudy or otherwise abnormal appearance should not be used.

Example 19 OptiFert Real-Time Fertility Assay Standard Operating Procedure v. 1.0 (February 2012) Process Control for Fertility Optimization 1.0 Intended Use

OptiFert is designed for use in process control during semen manufacture.

2.0 Introduction

In conventional semen processing, semen is collected, cooled and extended for packaging into straws, or is used immediately. In OptiFert processing, semen is collected, cooled, assayed by the OptiFert assay, and extended at a time that will enable maximum fertility using standard techniques such as IUI.

3.0 Principle

Sperm undergo a maturation process upon ejaculation. Sperm mature in groups, with fully activated sperm having a short life during which they will fertilize an egg, or they will die. OptiFert detects the presence of a biomarker, that appears during sperm maturation. This marker can be used to determine the maturational status of a group of sperm. With this knowledge, is possible to time the cooling period to biologically optimize a donation for fertility. OptiFert therefore optimizes fertility through use of mechanisms that already exist in nature, preserving sperm integrity.

4.0 Reagents and Materials

A collection cup, an assay kit and an SOP for use is supplied. The OptiFert assay reagents are supplied as three color-coded tubes (Green 1, Red 2 and Blue 3) plus a wash buffer (Buffer), and an SOP for use. Standard laboratory supplies are required. Scoring of the assay is by cytometry.

5.0 Safety and Handling Precautions

5.1 Safety Precautions

Follow standard safety procedures for handling semen as OPIM. Do not pipette by mouth. Do not smoke, eat or drink in areas in which samples or kit reagents are handled. Wash hands before leaving laboratory. Clean spills promptly. Dispose of materials properly. Sodium azide may react with lead or copper piping to form highly explosive metal azides. When disposing of liquid waste, dilute thoroughly to prevent the formation of such products.

5.2 Handling Precautions

Do not use kit components beyond their expiry date. Comply with instructions.

6.0 Sample Collection

The assay is performed directly on 5 ul aliquots of raw semen. Semen is collected into the OptiFert collection cup. Standard Operating Procedure (SOP) follows.

OptiFert Collection Cup Standard Operating Procedure Process Control for Fertility Optimization Safety

Follow standard procedures for handling of semen.

1. INSPECT COLLECTION CUP

-   -   a. Visually inspect cup for cracks or damage before using. Use         only cups that are intact.

2. BRING CUP TO OPERATING TEMPERATURE

-   -   a. Place cup in 32° C. water bath for at least 60 minutes. Make         sure cup is submerged in water up to the top of the insulated         section, so that the cup warms uniformly. Cups may be left in         bath overnight for use the next day.

3. OBTAIN DONATION AND BEGIN INCUBATION

-   -   a. Use standard methods of semen collection. Instruct donor to         place cup in cooling bath once sample is produced.     -   b. Keep cup submerged in water up to the top of the insulated         section during cooling period to ensure a smooth, controlled and         reproducible drop in the temperature of the ejaculate.

4. ASSAY

a. Follow instructions in the OptiFert Assay SOP for evaluating cells and carrying out further processing.

7.0 Assay Procedure

7.1 Equipment Required

Precision micropipettes or similar equipment with disposable tips for measurement of 5 ul, 10 ul, 20 ul, 100 ul and 1 ml are required. Calibration of these pipettes must be checked regularly. A calibrated cytometer suitable for use with the fluorophore fluorescein isothiocyanate (FITC) is needed. Also needed is a microfuge capable of accepting 1.5 ml snap-cap tubes, such tubes, and distilled water.

OptiFert Assay Standard Operating Procedure v 2.0 (March 2012) Process Control for Fertility Optimization Safety

Follow standard procedures for handling of semen. Before running this assay, be sure that semen is collected and incubated exactly as instructed in the OptiFert COLLECTION CUP SOP to minimize process failures.

1. PREPARE ASSAY MIXTURE

-   -   i. Into 1.5 ml tube, pipet the following IMMEDIATELY before use:     -   ii. 100 ul GREEN 1     -   iii. 20 ul RED 2     -   iv. 5 ul BLUE 3     -   v. 10 ul neat semen, mix.     -   vi. You will repeat this assay at 30 min intervals. Keep         reagents cool at all times.

2. INCUBATE

a. Place tube in dark for 20 minutes at ambient temperature

3. WASH

-   -   a. Add 1 ml BUFFER at ambient temperature     -   b. Microfuge 30 seconds     -   c. Carefully remove supernatant with 1 ml pipet.

4. SCORE

-   -   a. Add ˜200 ul BUFFER to cell pellet and mix gently to resuspend     -   b. Place tube containing resuspended cells into cytometer tube         and analyze on a calibrated cytometer using the “Cytometer         Scoring” template (see Cytometer Scoring SOP for further         details).

5. DETERMINE PROCESSING TIME FOR OPTIMAL FERTILITY

-   -   a. Plot percentage of positive cells. The percentage of cells         positive for the Biomarker will usually begin to increase in the         timeframe of 30 min-4 h post-collection. Time zero=time of assay         (which should be within less than 5 min post-collection).     -   b. When the peak of positivity of cells is seen, and desired         assay-based incubation time has been reached, semen should be         immediately stabilized at that desired time to maintain the         desired state. This can be accomplished in a number of ways,         often including extending semen with additives and         cooling/freezing. Additives should be introduced at 12 C, with         immediate transfer of extended collection to 4 C and subsequent         freezing. Alternatively, vitrification is also a suitable         method. Immediate use of semen for insemination is also a         method. Caution: deviation from recommended timeframe can         produce suboptimal results! You may extend semen and process         semen in the standard protocol in use at your facility, with the         following change: use extender cooled to 12oC before it is added         and begin further cooling immediately after extension, at the         time specified by assay result and desired outcome (e.g., good         fertility).         Definitions with respect to the SOPs disclosed herein:         The phrase “Green 1” as disclosed herein is Antibody Diluent         Solution, Cat #003118, Invitrogen/Life Technologies         The phrase “Red 2” as disclosed is rabbit polyclonal antiserum         directed to various antigens (for example, most often used is         against Listeria antigens, (but also Salmonella, Nesseria,         Meningitides, Campylobacter, among others), one example of         anti-Listeria antiserum would be Difco Listeria O antiserum type         4, Cat #223011, reconstituted with buffer (described below) to         the volume indicated on the lyophilized antiserum vial, Becton         Dickenson         Blue 3” as disclosed is Alexa Fluor 488 Goat anti-rabbit IgG         (H+L), 2 mg/ml, 6 mol dye/mole Cat # A11008,         Invitrogen/Molecular Probes/Life Technologies         The phrase “Buffer” as disclosed with respect to SOPs is: PBS         Tablets without calcium without magnesium, 1 tablet makes 100         ml, Cat #2810305, MP Biomedicals LLC.         OptiFert Cytometer Scoring—with Accuri C6

Standard Operating Procedure Process Control for Fertility Optimization Safety

Follow standard procedures for handling of semen. Before scoring this assay, be sure that semen is collected and incubated exactly as instructed and that the assay is run exactly as instructed, to minimize process failures.

1. START EQUIPMENT

-   -   i. Turn on computer     -   ii. Turn on cytometer (Accuri, Lansing, Mich.)     -   iii. If needed, empty waste bottle and fill sheath bottle with         DI water

2. OPEN TEMPLATE AND NAME FILE

-   -   a. Click OptiFert Template     -   b. Select File>Save CFlow file as . . .     -   c. Name file by date by typing date code under File Name     -   d. Click Save

3. COLLECT DATA

-   -   a. Under the red Collect tab, click on desired cytometer grid         (1A is for first sample, first donor, 1B is for second sample,         first donor. 2A is for first sample, second donor, etc.)     -   b. Next to cytometer grid (e.g., A01) type sample information         and assay time (time when collection is assayed) For example,         this would be written as Donor 9.30 for first time point from         donor providing a sample shortly before 9.30 am.     -   c. Check that stoplight shows green color. Load sample onto SIP         tube on cytometer and pull out plastic tube support underneath         tube     -   d. Click Run     -   e. After data are acquired, adjust gate on density plot so that         the percentage of positive sperm (the population on the right)         can be determined. Record this number. Remove sample from SIP         tube     -   f. For the next sample, repeat process above starting with step         3a     -   g. After the samples for that assay time are finished, click         Backflush, wait for stoplight to show green, then click Unclog

4. CLEAN AND SHUT DOWN EQUIPMENT

-   -   a. Between every sample, place towel under SIP, select Backflush         or Unclog     -   b. At the end of the day, place a tube of cleaning fluid on the         cytometer, select well H11 and click Run. Run for 2 minutes.         Then place a tube of water on the cytometer, select well H11 and         click Run. Run for 2 minutes. Allow water to clean system for at         least 2 minutes.     -   c. Turn off cytometer, then turn off computer         5. Maintenance—follow instructions in Accuri manual for         instrument cleaning. This should include every Wednesday and         Friday at day's end the extended cleaning of flow cell (found         under the Instrument tab on the cytometer menu).

8.0 Quality Control

A release QC test, designed for use on OptiFert frozen straws after thawing, is under development. The QC test will be used to confirm that the process has generated straws of optimized for fertility from the tested collections. We welcome inquiries about QC release test availability.

9. Results

To date, results with animal trials indicate that the process is capable of producing frozen straws of semen with improved fertility over conventionally-prepared straws from split bovine ejaculates.

10. Limitations

Assay reagents with cloudy or otherwise abnormal appearance should not be used. For atypical assay results, please contact Technical Service to determine if the results are acceptable for processing or if the collection is QC reject on assay results. Deviation from indicated times of stabilizing cells, once assay-based incubation time is reached, has produced unsatisfactory results in animal trials.

Example 19 Biomarker assay Process Standard Operating Procedure v. 1.8 (July 2011) Process Control for Fertility-preserving Gender Bias

1.0 Intended Use The biomarker assay process is designed for use in process control during semen manufacture.

2.0 Introduction

In conventional semen processing, semen is collected, cooled and extended for packaging into straws. In biomarker processing, semen is collected, cooled, assayed by the biomarker assay, and extended at a time that will enable production of a moderate female bias with good fertility upon AI of cows and heifers using standard techniques.

3.0 Principle

Sperm undergo a maturation process upon ejaculation. Sperm mature in groups, with fully activated sperm having a short life during which they will fertilize an egg, or they will die. The biomarker assay detects the presence of a marker that appears during sperm maturation. This marker can be used to determine the maturational status of a group of sperm. Knowledge of the maturation status of sperm is combined with another set of observations: (1) Y-bearing sperm, that fertilize to create males, mature and grow old slightly faster than X-bearing sperm and (2) X-sperm in a maturational group persist longer than Y-sperm. By knowing the maturational status of a sperm group, and that the earliest-maturing are Y-enriched while the longest-lasting are X-enriched, it is possible to time the cooling period to biologically enrich straws for X-bearing sperm, without a physical separation or sort of any type. Biomarker assay is therefore is a gentle treatment of sperm, producing moderate bias through use of natural mechanisms that already exist in nature. This preserves sperm integrity and enables AI of both heifers and cows under conditions that preserve fertility.

4.0 Reagents and Materials

A collection device, and an SOP for use is supplied. The biomarker assay reagents are supplied as three color-coded tubes (Green 1, Red 2 and Blue 3) plus a wash buffer (Buffer), and an SOP for use. Standard laboratory supplies are required. Scoring of the assay is by microscopy or cytometry.

5.0 Safety and Handling Precautions

5.1 Safety Precautions

Follow standard safety procedures for handling livestock and cattle semen. Do not pipette by mouth. Do not smoke, eat or drink in areas in which samples or kit reagents are handled. Wash hands before leaving laboratory. Clean spills promptly. Dispose of materials properly. Sodium azide may react with lead or copper piping to form highly explosive metal azides. When disposing of liquid waste, dilute thoroughly to prevent the formation of such products.

5.2 Handling Precautions

Do not use kit components beyond their expiry date. Comply with instructions.

6.0 Sample Collection

The assay is performed directly on 5 ul aliquots of raw semen. Semen is collected into the pre-warmed collection device by attaching the device to the artificial vagina (AV). Standard Operating Procedure (SOP) for the collection device follows.

Definitions: Green 1 is Antibody Diluent Solution, Cat #003118, Invitrogen/Life Technologies

Red 2 is rabbit polyclonal antiserum directed to various antigens (for example, most often used is against Listeria antigens, (but also Salmonella, Nesseria, Meningitides, Campylobacter, among others), one example of anti-Listeria antiserum would be Difco Listeria O antiserum type 4, Cat #223011, reconstituted with buffer (described below) to the volume indicated on the lyophyllized antiserum vial, Becton Dickenson Blue 3 is Alexa Fluor 488 Goat anti-rabbit IgG (H+L), 2 mg/ml, 6 mol dye/mole Cat # A11008, Invitrogen/Molecular Probes/Life Technologies Buffer is: PBS Tablets without calcium without magnesium, 1 tablet makes 100 ml, Cat #2810305, MP Biomedicals LLC.

Collection DEVICE Standard Operating Procedure Process Control for Fertility—Preserving Gender Bias Safety

Follow standard procedures for handling of semen.

1. INSPECT DEVICE AND INSERT BEAD

-   -   a. Visually inspect device for cracks or damage before using.         Use only devices that are intact.     -   b. Place one bead into each intact device.

2. BRING DEVICE TO OPERATING TEMPERATURE

-   -   a. Place device in 32oC water bath for at least 60 minutes. Make         sure device is submerged in water up to the cap of the large         tube, so the device warms uniformly. Devices may be left in bath         overnight for use the next day.

3. PERFORM COLLECTION AND BEGIN INCUBATION

-   -   a. Use standard methods of attachment to AV and of collection.         If device is out of water bath for more than 5 minutes between         placement onto AV and collection, remove it from AV and replace         with another device from the 32oC water bath, so that the         collection temperature will remain near 32oC.     -   b. Within 1 minute of collection, retrieve device, cap and         invert once, then place immediately into 12oC water bath.         Failure to carry out these steps quickly may result in process         failure.     -   c. Measure volume after tube has been in the 12oC bath for at         least 15 minutes, in order to minimize temperature changes. Keep         tube submerged in water up to the cap of the large tube during         the cooling period to ensure a smooth and uniform drop in the         temperature of the ejaculate.

4. ASSAY

-   -   a. Follow instructions in the Biomarker Assay SOP for evaluating         cells and carrying out further processing.

7.0 Assay Procedure

7.1 Equipment Required

Precision micropipettes or similar equipment with disposable tips for measurement of 5 ul, 10 ul, 20 ul, 100 ul and 1 ml are required. Calibration of these pipettes must be checked regularly. A calibrated cytometer or fluorescence microscope (research use only) suitable for use with the fluorophore fluorescein isothiocyanate (FITC) is needed. Also needed are a microfuge capable of accepting 1.5 ml snap-cap tubes, such tubes, and distilled water.

Biomarker Assay Standard Operating Procedure Process Control for Fertility—Preserving Gender Bias Safety

Follow standard procedures for handling of semen. If carrying out microscopy, do not look directly into the fluorescent light. Before running this assay, be sure that semen is collected incubated exactly as instructed in the collection device SOP to minimize process failures.

1. TREAT

-   -   i. Into 1.5 ml tube, pipet the following IMMEDIATELY before use:     -   ii. 100 ul GREEN 1     -   iii. 20 ul RED 2     -   iv. 5 ul BLUE 3     -   v. 5 ul neat semen, mix.     -   vi. Assay at 30 min intervals. Keep reagents cool at all times.

2. INCUBATE

-   -   a. Place tube in dark for 20 minutes at ambient temperature

3. WASH

-   -   a. Add 1 ml BUFFER at ambient temperature     -   b. Microfuge 30 seconds     -   c. Carefully remove supernatant with 1 ml pipet.

4. SCORE

-   -   a. Add ˜200 ul BUFFER to cell pellet and mix gently to resuspend     -   b. FOR MICROSCOPE (research use only): Transfer ˜5 ul to slide         and score # positive sperm (green fluorescence on head) and         #total cells. Count at least 100 cells. Calculate % positive. (%         Positive=[# positive/# total cells]×100)     -   c. FOR CYTOMETER: place aliquot of resuspended cells into         cytometer tube and analyze on a calibrated cytometer using the         “Biomarker Assay” template (see Assay Scoring SOP for further         details).

5. DETERMINE PROCESSING TIME

-   -   a. Plot percentage of positive cells. The percentage of cells         positive for the Biomarker will usually peak in the timeframe of         30 min-4 h post-collection. Time zero=time of collection.     -   b. Two hours (2 h) after the peak positivity of cells for the         Biomarker, semen should be extended. Caution: deviation from 2 h         can cause process failure! Extend semen and process semen in the         standard protocol in use at your AI station, with the following         change: use extender cooled to 12oC before it is added.         Biomarker Assay Scoring—with Accuri C6 Cytometer

Standard Operating Procedure Process Control for Fertility—Preserving Gender Bias Safety

Follow standard procedures for handling of semen. Before scoring this assay, be sure that semen is collected and incubated exactly as instructed and that the assay is run exactly as instructed, to minimize process failures.

1. START EQUIPMENT

-   -   i. Turn on computer     -   ii. Turn on cytometer (Accuri, Lansing, Mich.)     -   iii. If needed, empty waste bottle and fill sheath bottle with         DI water

2. OPEN TEMPLATE AND NAME FILE

-   -   a. Click Biomarker assay Template     -   b. Select File>Save CFlow file as . . .     -   c. Name file by date by typing date code under File Name     -   d. Click Save

3. COLLECT DATA

-   -   a. Under the red Collect tab, click on desired cytometer grid         (1A is for first sample, first bull, 1B is for second sample,         first bull. 2A is for first sample, second bull, etc.)     -   b. Next to cytometer grid (e.g., A01) type sample information         and batch time (time when all collections are batched for assay)         For example, this would be written as RDU 9.30 for first time         point from bull RDU)     -   c. Check that stoplight shows green color. Load sample onto SIP         tube on cytometer and pull out plastic tube support underneath         tube     -   d. Click Run     -   e. After data are acquired, adjust gate on density plot so that         the percentage of positive sperm (the population on the right)         can be determined. Record this number. Remove sample from SIP         tube     -   f. For the next sample, repeat process above starting with step         3a     -   g. After the samples for that hour are finished, click         Backflush, wait for stoplight to show green, then click Unclog

4. CLEAN AND SHUT DOWN EQUIPMENT

-   -   a. Between every sample, place towel under SIP, select Backflush         or Unclog     -   b. At the end of the day, place a tube of cleaning fluid on the         cytometer, select well H11 and click Run. Run for 2 minutes.         Then place a tube of water on the cytometer, select well H11 and         click Run. Run for 2 minutes. Allow water to clean system for at         least 2 minutes.     -   c. Turn off cytometer, then turn off computer         5. Maintenance—follow instructions in Accuri manual for         instrument cleaning. This should include every Wednesday and         Friday at day's end the extended cleaning of flow cell (found         under the Instrument tab on the cytometer menu).

8.0 Quality Control

A release QC test, designed for use on biomarker-processed straws after thawing, is under development. The QC test will be used to confirm that the biomarker process process has generated straws of semen enriched for viable X-sperm from the tested collections. We welcome inquiries about QC release test availability and can provide referrals to certified QC service laboratories.

9. Results

To date, results with this process indicate that the process is capable of production of a moderate female bias with preservation of fertility.

10. Limitations

Collections displaying abnormal quality at collection should not be processed. Abnormal morphology, excessively thin or otherwise abnormal ejaculates should not be used. Assay reagents with cloudy or otherwise abnormal appearance should not be used. For atypical assay results, please contact Technical Service to determine if the results are acceptable for processing or if the collection is QC reject on assay results.

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1. A method of optimizing sperm performance of a semen sample upon insemination of said semen sample, comprising: i) selecting a marker, wherein expression of the marker in the semen sample changes with time during capacitation; ii) determining the level or location of expression of the marker in the semen sample at a plurality of time points during incubation of said semen sample before insemination of said semen sample; iii) determining a timepoint for preparing said semen sample for use in insemination, wherein said timepoint is based upon a calibration of the marker expression displayed by said semen sample in step (ii) to a kinetic model of biomarker expression during sperm capacitation relative to said performance; iv) preparing at about said timepoint of step (iii) said semen sample for use in insemination; thereby optimizing said performance of said semen sample upon insemination of said semen sample.
 2. A method of optimizing sperm performance of a semen sample upon insemination of said semen sample, wherein said semen sample has been exposed to a treatment which modulates the rate of capacitation, comprising: i) selecting a marker, wherein expression of the marker in the semen sample changes with time during capacitation; ii) determining the level or location of expression of the marker in the semen sample at a plurality of time points after exposure to said treatment and during incubation of said semen sample before insemination of said semen sample, iii) determining a timepoint for preparing said semen sample for use in insemination, wherein said timepoint is based upon a calibration of the marker expression displayed by said semen sample in step (ii) to a kinetic model of biomarker expression during sperm capacitation relative to said performance; iv) preparing at about said timepoint of step (iii) said semen sample for use in insemination; thereby optimizing said performance of said semen sample upon insemination of said semen sample.
 3. The method of claim 1 or 2, wherein the semen sample is mammalian.
 4. The method of claim 1 or 2, wherein the semen sample is human.
 5. The method of claim 1 or 2, wherein the sperm performance comprises enhanced fertility.
 6. The method of claim 1 or 2, wherein the sperm performance comprises gender bias.
 7. The method of claim 1 or 2, wherein the sperm performance comprises female gender bias.
 8. The method of claim 1 or 2, wherein the sperm performance comprises male gender bias.
 9. The method of claim 1 or 2, wherein preparing said semen sample for use in insemination comprises freezing said semen sample or vitrification of said semen sample.
 10. The method of claim 9, wherein the sperm performance comprises resistance to freezing when preparing said semen sample for use in insemination.
 11. The method of claim 9, wherein the sperm performance comprises resistance to vitrification when preparing said semen sample for use in insemination.
 12. The method of claim 1 or 2, wherein the marker and the biomarker are identical.
 13. The method of claim 1 or 2, wherein the marker and/or the biomarker is an Fc receptor.
 14. The method of claim 1 or 2, further comprising more than one marker.
 15. The method of claim 1 or 2, wherein the kinetic model of biomarker expression during sperm capacitation comprises more than one biomarker.
 16. The method of claim 1 or 2, wherein the kinetic model of biomarker expression during sperm capacitation comprises a biomarker which binds to the constant region of an antibody.
 17. The method of claim 1 or 2, wherein the semen sample is not treated by fixation.
 18. The method of claim 1 or 2, wherein the semen sample is not exposed to treatment to increase permeability of the membrane.
 19. The method of claim 1 or 2, wherein the first of said plurality of time points is obtained immediately after collection of said semen sample.
 20. The method of claim 1 or 2, wherein the marker and the biomarker are identical.
 21. The method of claim 1 or 2, wherein the marker and the biomarker is an Fc receptor.
 22. The method of claim 1 or 2, further comprising more than one marker.
 23. The method of claim 1 or 2, wherein the kinetic model of biomarker expression during sperm capacitation comprises more than one biomarker.
 24. The method of claim 1 or 2, wherein the kinetic model of biomarker expression during sperm capacitation comprises the biomarker Fc receptor.
 25. The method of claim 1 or 2, wherein the kinetic model of biomarker expression during sperm capacitation is developed using ejaculates from the same species of said semen sample.
 26. The method of claim 1 or 2, wherein said incubation of said semen sample comprises incubation at a temperature ranging from approximately 40 C to 4° C.
 27. The method of claim 26, wherein said incubation of said semen sample occurs at a temperature gradient ranging from approximately 40 C to 12° C.
 28. The method of claim 2, wherein said treatment decelerates the rate of capacitation.
 29. The method of claim 2, wherein said treatment accelerates the rate of capacitation.
 30. The method of claim 2, wherein said treatment arrests capacitation at a specific stage.
 31. The method of claim 2, wherein said treatment comprises exposing said semen sample to a chemical agent which modulates the rate of capacitation.
 32. The method of claim 31, wherein said agent is selected from the group consisting of endocannibinoids, bicarbonate and 8-butyryl cAMP.
 33. The method of claim 2, wherein said treatment comprises exposing said semen sample to an environmental stimulus which modulates the rate of capacitation.
 34. The method of claim 33, wherein said environmental stimulus is selected from the group consisting of barometric pressure, atmospheric pressure, temperature change and agitation.
 35. The method of claim 33, wherein said treatment comprises exposing said semen sample to an atmospheric condition which modulates the rate of capacitation.
 36. The method of claim 35, wherein said atmospheric condition comprises a gas which is selected from the group consisting of a nitric oxide, ozone, argon, cyanide and CO₂.
 37. The method of claim 36, wherein said the CO₂ concentration is greater than 5%.
 38. The method of claim 1 or 2, wherein the marker or biomarker is expressed in the extracellular portion of said semen sample.
 39. The method of claim 1 or 2, wherein the marker or biomarker is expressed by a sperm cell.
 40. The method of claim 39, wherein the marker or biomarker is expressed on the cell surface of said sperm cell.
 41. The method of claim 39, wherein the marker or biomarker is expressed in a membrane of the sperm cell.
 42. The method of claim 41, wherein the marker or biomarker is a change selected from the group consisting of membrane isotrophy, membrane fluidity, membrane charge, membrane permeability, membrane budding, membrane hydrophobicity, lipid raft structure, a lipid subdomain of the membrane, ion channel permeability, tyrosine phosphorylation, kinase activation, protease activation, kinase inactivation, protease inactivation, change in presence or amount of a ligand-binding molecule(s), and a calcium gradient.
 43. The method of claim 39, wherein the marker or biomarker is a lipid selected from the group of phosphatidyl serine, phosphatidlycholine, phosphatidyl ethanolamine and sphingomyelin.
 44. The method of claim 39, wherein the marker or biomarker further comprises its cellular location of the sperm.
 45. The method of claim 39, wherein the marker or biomarker is intracellularly expressed with respect to said sperm cell.
 46. The method of claim 45, wherein the intracellular marker or biomarker is selected from the group consisting of: intracellular pH, intracellular concentration of HCO₃, intracellular concentration of fragmented DNA, mitochondrial Calcium and intracellular concentration of Calcium.
 47. The method of claim 39 where the marker or biomarker binds a glycoprotein or an anionic polysaccharide.
 48. The method of claim 39, where the marker or biomarker binds a glycosaminoglycan, a sulfated glycosaminoglycan, a sulfated glycan and a sulfated polylactosaminoglycans.
 49. The method of claim 39, where the marker or biomarker is selected from the group consisting of heparin, fucoidan, ZP3, a glycoprotein or fragment thereof derived from the egg vestments, and a Lewis antigen.
 50. The method of claim 39, where the marker or biomarker binds a dye.
 51. The method of claim 39, wherein the marker or biomarker is a physiologic activity selected from the group consisting of percent of sperm displaying motility, motility grade, frequency of beating of flagella of said sperm cell, ability of said sperm cell to penetrate mucus, loss of adhesion of said sperm cell, hypoosmotic swelling of said sperm cell, chemotaxis, thermotaxis and metabolic status of said sperm cell.
 52. The method of claim 39, where the marker or biomarker comprises a molecule secreted or expelled by said sperm cell.
 53. The method of claim 38, wherein the marker or biomarker comprises the appearance of exocytic vesicles or hybrid vesicles in solution.
 54. The method of claim 52, where in the secreted or expelled molecule is selected from the group consisting of an enzyme, a proenzyme, an agent, a reactive oxygen species, an exosomal vesicle, a dye and DNA fragments.
 55. The method of claim 52, where in the secreted or expelled agent is selected from the group consisting of an antibody bound fluorophore, a fluorophore and an enzyme.
 56. The method of claim 1 or 2, wherein said biomarker is selected from the group consisting of Fertility-associated antigen, soybean trypsin inhibitor, an Fc receptor and CD46.
 57. The method of claim 1 or 2, wherein said marker is selected from the group consisting of Fertility-associated antigen, soybean trypsin inhibitor, an Fc receptor and CD46.
 58. The method according to claim 9, wherein freezing said semen sample comprises adding a cryoprotectant selected from the group consisting of trehalose, glycerol, propylene glycol, dimethylsulfoxide, sucrose and egg yolk.
 59. The method of claim 39, where the marker or biomarker is permeability of said sperm to a dye.
 60. The method of claim 59, where the marker or biomarker is oscillatory behavior of said sperm in response to dye uptake and/or release.
 61. The method of claim 49, wherein the egg vestments of fragment thereof, is from the zona pellicida or fragments thereof. 